Embryonic malformations in an offspring of the shortnose guitarfish

Embryonic malformations can be a result of exposure toxic substances (pollution), lack of nutrients, physical restraint, genetic disorders, or infections and diseases. Such malformations can be classified according to severity and offspring survival outside the uterus. Moreover, abnormalities are normally restricted to a small portion of the litter, mostly, to only one embryo. Here, we report a case of congenital malformation dominance in an offspring of the shortnose guitarfish and compare it with other abnormalities commonly reported in batoids in addition to discussing the possible causes involved in the observed deformities. AbstrAct Natascha Wosnick1, Verônica Takatsuka2, Ana Eliza Mello3, June Dias4, Nicolas Lubitz5, Venâncio Guedes de Azevedo2*

A teratogen is any environmental, infectious or nutritional agent capable of producing developmental disturbances or congenital malformations in an embryo after maternal exposure to it (Mahan and Escott-Stump, 2003). A fundamental difference between a malformation caused by a mutation and one resulting from a teratogenic agent is that, in the latter case, there is no change in the genetic load. Consequently, there is no risk of transmission to offspring when the affected individual reproduces. Despite knowledge that a teratogenic agent can lead to congenic anomalies in several organisms, the critical stage of development in which exposure must occur so that the effects are manifested remains unknown. However, it is known that the main mechanisms by which a teratogen acts are inhibition of cell proliferation and protein synthesis, generalized cell death and changes in differentiation patterns and morphogenic interactions (Shepard et al., 1975).
To date, congenital anomalies have been described only for viviparous species, given the greater maternalfetal interaction in this reproductive mode. However, occurrence is not restricted to matrotrophy, since reports in lecithotrophic species are also common (Ribeiro-Prado et al., 2008;Bornatowski and Abilhoa, 2009;Carmo and Fávaro, 2015). Zapteryx brevirostris (Müller and Henle, 1841) is a small-sized guitarfish, endemic to the Southwest Atlantic, with a lecithotrophic viviparous mode and up to 6 embryos per litter (Figueiredo, 1977;Batista, 1991). In Southern Brazil, the embryonic development period occurs during spring, with birth occurring in warmer months (December to March) (Batista, 1991;Abilhoa et al., 2007). Reports on Z. brevirostris malformations are recent, with only two occurrences described in the literature, one related to an embryo malformation (one in a litter of five embryos) (Carmo and Fávaro, 2015) and another being a case of anophthalmia in an adult female (Ribeiro-Prado et al., 2008). Here, we report for the first time an occurrence of congenital malformation in multiple embryos of the same offspring, compare them with abnormalities commonly found in other elasmobranchs and discuss possible causes for the alterations observed.
One adult female was captured as bycatch of shrimp trawling on the northern coast of São Paulo (SISBIO 49980-3 and Ethics Committee on Animal Use of the Fisheries Institute -CEEAIP 12 / 2016). The female was caught in the last haul of the trawl and transferred to a 200L styrofoam box, with constant aeration and water circulation. Afterwards, it was transported to the laboratory facilities and acclimatized in a 2500L glass fiber tank. Food was offered once a day in a satiety feeding regime, consisting of beheaded Atlantic seabob shrimp, Xiphopenaeus kroyeri (Heller, 1862). After acclimation, the female, along with other individuals of the same species, was used in experiments aiming to evaluate the influence of different sediments on behavior and physiological performance (unpublished data). After the experimental period (18 days), the specimen was euthanized through exposure to high dosage of anesthetic (eugenol diluted in alcohol 15-168 mg/L) for the assessment of systemic condition.
During necropsy, pregnancy was detected. Five embryos were present and four of them exhibited anatomical malformations. Internal observations were made through x-rays, taken by CDK trigger and an Agfa CR 10 digitizer with the following configurations: KV 40 and mAs of 6.4.
The embryos were held in dorsal position for diagnosis. All embryos were carefully evaluated, and the description of the deformities is given below.
Specimen one: mild malformations; absence of the final portion of the tail; no signs of fracture; X-ray diagnosis revealed no formation of the final portion of the tail (total length -6.9cm, disc width -3.9cm, weight -4g) ( Figure  1a). Specimen two: mild malformations; fins not fully fused in the lateral portions of the tail base; X-ray diagnosis revealed a midline malformation in the final portion of the tail (total length -7.9cm, disc width -3.5cm, weight -2.1g) (Figure 1b). Specimen three: severe malformations; both pectoral fins not fully fused in the cephalic region; X-ray analysis diagnosis revealed midline deformation at the last portion of the tail, a mild midline deviation in the pelvic girdle region and fused gills (total length -7.8cm, disc width -3.2cm, weight -2.6g) (Figure 1c). Specimen four: severe malformations; pectoral fins not fully fused in the cephalic region, with a more pronounced deformation in the right pectoral fin; malformation of both pelvic fins; severe midline deviation in the pelvic girdle region that extended to the entire caudal region (total length -8cm, disc width -3.8cm, weight -3g) (Figure 1d). Specimen five: fully formed, with no signs of external or internal abnormalities (total length -9.2cm, disc width -4cm, weight -4.4g) (Figure 1e). Given the birth size of the species (~ 14cm in TL - Batista, 1991), it was estimated that female was in the third trimester of pregnancy (12 months in the species - Batista, 1991).
The abnormalities observed in the present study were related to morphological alterations of the pectoral fins and spinal malformation. In a recent survey, some occurrence patterns were observed, with females being more affected than males, and malformations in the cephalic region (chondrocranium deformation) and pelvic fins being the most commonly observed in batoids and sharks (Nunes et al., in prep). Still, studies show that spinal deformations such as lordosis, scoliosis and kyphosis are also common but less deleterious (Berzins et al., 2002;Preziosi et al., 2006;Pérez et al., 2018), with lower mortality rates. Nonetheless this may be associated with negative consequences, as swimming capacity may be hampered (Muñoz-Osorio, et al., 2013;dos Santos & Gadig, 2014).
The offspring of the present study indeed presented the most commonly observed deformities in elasmobranchs, however at a frequency never reported in the literature, since 80% of embryos had morphological alterations. Such a phenomenon, although occurring in elasmobranchs that have more than one developing embryo at the same time, appears to have low occurrence in the same offspring, with usually only one or two embryos exhibiting significant morphological alterations (Bornatowski and Abilhoa, 2009;dos Santos and Gadig, 2014;Carmo and Fávaro, 2015;Lamarca et al., 2017). This fact is of significance, since all embryos are exposed to the same uterine conditions and maternal-fetal interactions, thus future studies aiming to understand why malformations are not prevalent within an offspring should be carried out.
Since in the present study most of the offspring presented significant morphological alterations, it is possible that the phenomenon was related to exposure to a teratogen in the initial stages of embryonic development. In fact, there is strong evidence that the most serious congenital malformations are related to exposure during the critical period of differentiation and organogenesis, since at this stage the embryo is more vulnerable to changes at the structural level (Nelson, 1957). Also, among the teratogenic agents mentioned above, it is possible that the malformations reported are related to female starvation and/or severe nutritional deficiency in the wild rather than exposure to environmental contaminants, due to the predominance observed. Such inference is based on a study performed with pregnant round stingrays (Urobatis helleri) (Cooper, 1863) exposed to contaminants, with significant alterations on female's metabolism, but no embryonic malformation (Lyons, 2018).
In summary, here we present the first report of embryonic malformation predominance in an elasmobranch. Although the causes are reason for speculation, such occurrence is extremely significant as it increases the knowledge about congenital malformations in this group, as well as offering new perspectives in terms of the frequency of occurrence.