Evolution and Diversity of TGF-ß Pathways are Linked with Novel Developmental and Behavioral Traits.

Transforming growth factor-ß (TGF-ß) signaling is essential for numerous biologic functions. It is a highly conserved pathway found in all metazoans including the nematode Caenorhabditis elegans, which has also been pivotal in identifying many components. Utilizing a comparative evolutionary approach, we explored TGF-ß signaling in nine nematode species and revealed striking variability in TGF-ß gene frequency across the lineage. Of the species analyzed, gene duplications in the DAF-7 pathway appear common with the greatest disparity observed in Pristionchus pacificus. Specifically, multiple paralogues of daf-3, daf-4 and daf-7 were detected. To investigate this additional diversity, we induced mutations in 22 TGF-ß components and generated corresponding double, triple, and quadruple mutants revealing both conservation and diversification in function. Although the DBL-1 pathway regulating body morphology appears highly conserved, the DAF-7 pathway exhibits functional divergence, notably in some aspects of dauer formation. Furthermore, the formation of the phenotypically plastic mouth in P. pacificus is partially influenced through TGF-ß with the strongest effect in Ppa-tag-68. This appears important for numerous processes in P. pacificus but has no known function in C. elegans. Finally, we observe behavioral differences in TGF-ß mutants including in chemosensation and the establishment of the P. pacificus kin-recognition signal. Thus, TGF-ß signaling in nematodes represents a stochastic genetic network capable of generating novel functions through the duplication and deletion of associated genes.

Influence of environmental temperature on mouth-form plasticity in Pristionchus pacificus acts through daf-11-dependent cGMP signaling.

Mouth-form plasticity in the nematode Pristionchus pacificus has become a powerful system to identify the genetic and molecular mechanisms associated with developmental (phenotypic) plasticity. In particular, the identification of developmental switch genes that can sense environmental stimuli and reprogram developmental processes has confirmed long-standing evolutionary theory. However, how these genes are involved in the direct sensing of the environment, or if the switch genes act downstream of another, primary environmental sensing mechanism, remains currently unknown. Here, we study the influence of environmental temperature on mouth-form plasticity. We find that environmental temperature does influence mouth-form plasticity in most of the 10 wild isolates of P. pacificus tested in this study. We used one of these strains, P. pacificus RSA635, for detailed molecular analysis. Using forward and reverse genetic technology including CRISPR/Cas9, we show that mutations in the guanylyl cyclase Ppa-daf-11, the Ppa-daf-25/AnkMy2, and the cyclic nucleotide-gated channel Ppa-tax-2 eliminate the response to elevated temperatures. Together, our study indicates that DAF-11, DAF-25, and TAX-2 have been co-opted for environmental sensing during mouth-form plasticity regulation in P. pacificus.

The cGMP signaling pathway affects feeding behavior in the necromenic nematode Pristionchus pacificus.

BACKGROUND: The genetic tractability and the species-specific association with beetles make the nematode Pristionchus pacificus an exciting emerging model organism for comparative studies in development and behavior. P. pacificus differs from Caenorhabditis elegans (a bacterial feeder) by its buccal teeth and the lack of pharyngeal grinders, but almost nothing is known about which genes coordinate P. pacificus feeding behaviors, such as pharyngeal pumping rate, locomotion, and fat storage. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed P. pacificus pharyngeal pumping rate and locomotion behavior on and off food, as well as on different species of bacteria (Escherichia coli, Bacillus subtilis, and Caulobacter crescentus). We found that the cGMP-dependent protein kinase G (PKG) Ppa-EGL-4 in P. pacificus plays an important role in regulating the pumping rate, mouth form dimorphism, the duration of forward locomotion, and the amount of fat stored in intestine. In addition, Ppa-EGL-4 interacts with Ppa-OBI-1, a recently identified protein involved in chemosensation, to influence feeding and locomotion behavior. We also found that C. crescentus NA1000 increased pharyngeal pumping as well as fat storage in P. pacificus. CONCLUSIONS: The PKG EGL-4 has conserved functions in regulating feeding behavior in both C. elegans and P. pacificus nematodes. The Ppa-EGL-4 also has been co-opted during evolution to regulate P. pacificus mouth form dimorphism that indirectly affect pharyngeal pumping rate. Specifically, the lack of Ppa-EGL-4 function increases pharyngeal pumping, time spent in forward locomotion, and fat storage, in part as a result of higher food intake. Ppa-OBI-1 functions upstream or parallel to Ppa-EGL-4. The beetle-associated omnivorous P. pacificus respond differently to changes in food state and food quality compared to the exclusively bacteriovorous C. elegans.

A new fabrication process for a microchip electrophoresis device integrated with a three-electrode electrochemical detector.

We report here a novel and simple process for the fabrication of a poly(methyl methacrylate) (PMMA)-based microchip electrophoresis device, integrated with a screen-printed three-electrode electrochemical detector that does not require a replicate mold. In this approach, a photoresist layer constitutes both an adhesion layer and side walls of 50 mum wide and 50 mum tall microfluidic channels on a screen-printed three-electrode PMMA substrate. Openings were drilled for buffer reservoirs on an additional piece of PMMA, then the final device was bonded in a PMMA/photoresist/PMMA sandwich configuration. This process is inexpensive, less time-consuming, and simpler compared with traditional fabrication methods. The combination of this PMMA-based microchip fabrication together with screen-printed electrode technology holds great promise for the mass production of a single-use micrototal analytical system. Successful determination of uric acid and L-ascorbic acid with the presented system validates its utility. In combination with a suitable electrochemical detector, this device holds much promise for the determination of other analytes in various biological samples for medical and clinical diagnosis.