Experimental Crossing Confirms Reproductive Isolation between Cryptic Species within <i>Eulimnogammarus verrucosus</i> (Crustacea: Amphipoda) from Lake Baikal

Ancient lakes are known speciation hotspots. One of the most speciose groups in the ancient Lake Baikal are gammaroid amphipods (Crustacea: Amphipoda: Gammaroidea). There are over 350 morphological species and subspecies of amphipods in Baikal, but the extent of cryptic variation is still unclear. One of the most common species in the littoral zone of the lake, Eulimnogammarus verrucosus (Gerstfeldt, 1858), was recently found to comprise at least three (pseudo)cryptic species based on molecular data. Here, we further explored these species by analyzing their mitogenome-based phylogeny, genome sizes with flow cytometry, and their reproductive compatibility. We found divergent times of millions of years and different genome sizes in the three species (6.1, 6.9 and 8 pg), further confirming their genetic separation. Experimental crossing of the western and southern species, which are morphologically indistinguishable and have adjacent ranges, showed their separation with a post-zygotic reproductive barrier, as hybrid embryos stopped developing roughly at the onset of gastrulation. Thus, the previously applied barcoding approach effectively indicated the separate biological species within E. verrucosus. These results provide new data for investigating genome evolution and highlight the need for precise tracking of the sample origin in any studies in this morphospecies

The level of putative carotenoid-binding proteins determines the body color in two species of endemic Lake Baikal amphipods

Color is an essential clue for intra- and interspecies communication, playing a role in selection and speciation. Coloration can be based on nanostructures and pigments; carotenoids and carotenoproteins are among the most widespread pigments in animals. Over 350 species and subspecies of amphipods (Crustacea: Amphipoda) endemic to Lake Baikal exhibit an impressive variability of colors and coloration patterns, including intraspecific color morphs. However, the mechanisms forming this diversity are underexplored, as while the carotenoid composition of several transparent, green, and red species was investigated, there have been no reports on the corresponding carotenoid-binding proteins. In this work, we analyze the coloration of two brightly colored Baikal amphipods characterized by intraspecific color variability, Eulimnogammarus cyaneus and E. vittatus. We showed that the color of either species is defined by the level of putative carotenoid-binding proteins similar to the pheromone/odorant-binding protein family, as the concentration of these putative crustacyanin analogs was higher in blue or teal-colored animals than in the orange- or yellow-colored ones. At the same time, the color did not depend on the total carotenoid content, as it was similar between animals of contrasting color morphs. By exploring the diversity of these sequences within a larger phylogeny of invertebrate crustacyanins, we show that amphipods lack orthologs of the well-studied crustacyanins A and C, even though they possess some crustacyanin-like sequences. The analysis of expression levels in E. cyaneus showed that the transcripts encoding crustacyanin analogs had much higher expression than the crustacyanin-like sequences, suggesting that the former indeed contribute to the color of these brightly colored animals. The crustacyanin analogs seem to act in a similar way to the well-studied crustacyanins in body color formation, but the details of their action are still to be revealed

Turn a Shrimp into a Firefly: Monitoring Tissue pH in Small Crustaceans Using an Injectable Hydrogel Sensor with Infrared Excitation and Visible Luminescence

Various implantable optical sensors are an emerging tool in animal physiology and medicine that may provide real-time information about body fluids without tissue extraction. Such sensors are often fluorescence-based and require strong visible external illumination during signal acquisition, which causes anxiety or even stress for small animals and thus may influence the physiological parameters being measured. In order to overcome this obstacle, here, we combined a fluorescent molecular pH probe with upconverting particles within a hydrogel fiber suitable for injection into small crustaceans. The green luminescence of the particles under non-visible infrared illumination excited fluorescence of the molecular probe and allowed for pH measurements after correction of the probe readout for luminescence intensity. The developed optical setup based on a common microscope ensured effective visualization of the sensor and spectral pH measurements through the translucent exoskeleton of the amphipod (Amphipoda, Crustacea) Eulimnogammarus verrucosus, endemic to ancient Lake Baikal. Testing the sensors in these cold-loving crustaceans under environmentally relevant temperature increases showed alkalization of amphipod internal media by 0.2 soon after the start of the experiment, while further increases led to acidification by 0.5. The applied approach for simple sensor preparation can be useful in building other implantable optical sensors for light-sensitive organisms