Mesobiotus insanis, Mapalo & Stec & Mirano-Bascos & Michalczyk, 2017

Mapalo, Marc A., Stec, Daniel, Mirano-Bascos, Denise & Michalczyk, Łukasz, 2017, An integrative description of a limnoterrestrial tardigrade from the Philippines, Mesobiotus insanis, new species (Eutardigrada: Macrobiotidae: harmsworthi group), Raffles Bulletin of Zoology 65, pp. 440-454 : 442-445

Mesobiotus insanis new species

( Figs. 1–8 )

Material examined. Holotype (slide number: PH.003.12), 29 paratypes (slide numbers: PH.003.03–04, 06, 08–13, 15–16, 20), and 29 eggs (slide numbers: PH.003.07, 22, PH.004.01– 03; 6 eggs on SEM stubs) as well as 7 females processed for DNA sequencing. All examined individuals and eggs were derived from a single female isolated from moss on the trunk of a tree growing in the UP Science Park, near the College of Science Library , University of the Philippines, Diliman , Quezon City , Philippines (14°38′56.2″N, 121°04′10.3″E, 60 m asl). Coll. Marc Mapalo, Mika Berza and Cali Fernandez, May 2014.

Description of the new species. Animals (morphometrics in Table 4): Body white/transparent ( Fig. 1A ). Eyes absent in live individuals. Cuticle without pores. Granulation present on legs I–IV ( Fig. 1B, C , empty arrowheads).

Buccal apparatus of Macrobiotus- type with ventral lamina and ten peribuccal lamellae ( Fig. 2A ). Oral cavity armature of the harmsworthi type, composed of three bands of teeth visible under PCM ( Fig. 2 ). The first band of teeth appears as small granules arranged in several irregular rows along the anterior portion of the oral cavity and sometimes on the bases of the lamellae ( Fig. 2B–E , filled arrowheads). Teeth in the second band appear as small ridges that are parallel to the main axis of the buccal tube and situated at the posterior portion of the oral cavity ( Fig. 2B–E , empty arrowheads). Situated right after the second band of teeth and before the buccal tube opening is the third band of teeth ( Fig. 2B–E , indented arrowheads), consisting of 3 dorsal thin ridge teeth and 5–7 ventral teeth: 2 drop-shaped lateral teeth and 3–5 oval median teeth sometimes with additional teeth ( Fig. 2B–E , arrow). Buccal tube rigid, with a ventral lamina and a thickening posterior to the stylet support insertion points. Pharyngeal apophyses, three macroplacoids and a microplacoid present in the muscle pharynx. All placoids equidistant from each other. In the ventral view, the first and the third macroplacoid are rod-shaped whereas the second macroplacoid is drop-like. The first macroplacoid is thinner anteriorly whereas the third macroplacoid has a sub-terminal constriction, which is visible in both the ventral and the dorsal view ( Fig. 2F, G , empty indented arrowheads). The macroplacoid sequence is 2<1<3. The drop-like microplacoid is typically longer than the second macroplacoid (93% of the analysed animals, in the remaining 7% the second macroplacoid is equal to or slightly longer than the microplacoid).

Claws of the Mesobiotus type, with a peduncle connecting the claw to the lunula, a basal septum and well-developed accessory points on the primary branches ( Fig. 3A–C ). Lunules smooth under claws I–III ( Fig. 3A, B ) and slightly crenulated under claws IV ( Fig. 3C ). Single transverse W-shaped bars below claws I–III present ( Fig. 3B , arrow), whereas a horseshoe-shaped structure connects the anterior and posterior lunules on claws IV ( Figs. 1C , 3C , filled arrowheads).

Eggs (morphometrics in Table 5): Spherical, white, laid freely, with hemispherical to conical processes. The processes are equidistant from each other and vary in shape from low domes to high cones ( Fig. 6A–L ). The process surface is wrinkled and the wrinkles form a whorl that is clearly visible with SEM ( Fig. 8A–F ) but only appears as serrations on intersected process walls under PCM ( Fig. 6A–H ). Some eggs have smooth process surfaces under PCM ( Fig. 6I–L ). The labyrinthine layer within the process walls appears as reticulation under PCM, with meshes that vary in diameter considerably between eggs ( Fig. 7A–D ). A few scattered pores, especially in the basal portion of the process, are present in the external process wall (only clearly visible in SEM since in PCM the pores blend with the labyrinthine layer). Processes are terminated by at least 15 short, thin, and flexible filaments that are visible in both PCM ( Fig. 6A–L ) and SEM ( Fig. 8A–F ). The filaments are covered with tiny granules that are visible only in SEM ( Fig. 8D, E ). Processes are connected by 10–14 wide stripes ( Figs. 7 , 8A ) with smooth surfaces in SEM ( Fig. 8 ) but in PCM the stripes appear covered with mesh ( Fig. 7 ) and sometimes large bubbles ( Fig. 7D , empty arrowhead), which are all the representations of the labyrinthine layer below the surface of the stripes. The spaces between the connective stripes form single, complexly sculptured areolae on the egg surface (10–14 areolae around each process; Figs. 7 , 8A–D, F ). Each areola consists of a central rose-shaped whorl of fine

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Initial Annealing Elongation Final wrinkles ( Figs. 7A–C , 8C–F ) and porous surface around the whorl ( Fig. 8D, F ). Both whorls and pores are always clearly visible in SEM ( Fig. 8C–F ), however under PCM pores are never identifiable (they are below the resolution of light microscope) whereas whorls are visible only in some eggs ( Fig. 7A–C ).

Remarks. Eggs of the new species exhibit considerable variation in chorion morphology ( Figs. 4–8 ). Given that the eggs were obtained in culture, it should be considered whether similar variability could be observed in the wild. The great majority of eutardigrade species descriptions provide images of only most typical eggs morphotypes, which may result in a conviction that eutardigrade eggs are characterised by low intra-specific variability. However, recent studies show that at least in some eutardigrade species, egg chorion may exhibit considerable morphological and morphometric variation in natural populations ( Stec et al., 2017; Zawierucha et al., 2016). Moreover, eggs of R. subanomalus obtained by Stec et al. (2016b) in a laboratory culture showed the same extent of variation as eggs of the same species extracted from a moss sample by Stec et al. (2017), which suggests that M. insanis , new species, also may lay variable eggs in nature. Nevertheless, even if laboratory conditions indeed increased egg shell variability, the narrower natural variation would fall within that observed in the in vitro culture. In other words, a greater variability presented in the description would allow an easier identification of the species in the future.

DNA Sequences. A single haplotype was found for each of the four sequenced markers in all seven analysed individuals representing the three subcultures. The sequences were deposited in GenBank with the following reference numbers: 18S rRNA, 499 bp long, MF441488 ; 28S rRNA, 769 bp long, MF441489 ; ITS -2, 352 bp long, MF441490 ; COI, 771 bp long, MF441491 .

Etymology. The name of the new species refers to its insanely complex egg morphology, never observed before in any other tardigrade species.

Figures

Fig. 1. Mesobiotus insanis, new species, PCM images of habitus and cuticle. A, dorso-ventral projection (holotype, PCM); B, dorsal cuticle above leg II; C, ventral cuticle of legs IV. Empty arrowheads indicate granulation; the filled arrowhead indicates the horse-shoe shaped structure connecting the anterior and the posterior claw. Scale bars in μm.

Fig. 2. Mesobiotus insanis, new species, PCM images of the buccal apparatus. A, an entire buccal apparatus (paratype); B, dorsal teeth; C–E, ventral teeth (in Fig C, D, E, the medio-ventral tooth is divided into three, four, and five oval teeth, respectively); F, ventral placoids; G, dorsal placoids. Filled flat arrowheads indicate the first band of teeth, empty flat arrowheads indicate the second band of teeth, filled indented arrowheads indicate the third band of teeth, empty arrows indicate the additional teeth in the third band, empty indented arrowheads indicate subterminal constrictions in the third macroplacoid. Scale bars in μm.

Fig. 3. Mesobiotus insanis, new species, PCM images of claws. A, claws I with smooth lunules; B, claws II with smooth lunules; C, claws IV with slightly crenulated lunules. Arrows indicate the W-shaped cuticular bars, the filled arrowhead indicates the horseshoe-shaped structure connecting the anterior and the posterior claw, the empty arrowheads indicate leg granulation. Scale in μm.

Fig. 4. Mesobiotus insanis, new species, PCM images of midsections of six different eggs. Scale bars in μm.

Fig. 5. Mesobiotus insanis, new species, PCM images of the surfaces of the eggs shown in Fig. 4 (respective letters indicate same eggs in both figures). Scale bars in μm.

Fig. 6. Mesobiotus insanis, new species, PCM images of the midsection of processes from various eggs. Scale bars in μm.

Fig. 7. Mesobiotus insanis, new species, magnified PCM images of the surface of the eggs shown in Fig. 4 (respective letters indicate same eggs in both figures). Filled arrowheads indicate the whorled sculpturing inside areolae; empty arrowheads indicate the bubble-like structures within the rims delimiting the areolae. Scale bars in μm.

Fig. 8. Mesobiotus insanis, new species, SEM images of eggs. A, B, entire view of two different eggs; C, D, egg surface with processes; E, egg process; F, egg surface between processes, with evident pores and whorl-shaped sculpturing. Arrows indicate pores in the processes, flat arrowheads indicate the whorl-shaped surface inside the areolae, indented arrowheads indicate pores inside the areolae. Scale bars in μm.