High-throughput mass spectrometry analysis revealed a role for glucosamine in potentiating recovery following desiccation stress in Chironomus

Desiccation tolerance is an essential survival trait, especially in tropical aquatic organisms that are vulnerable to severe challenges posed by hydroperiodicity patterns in their habitats, characterized by dehydration-rehydration cycles. Here, we report a novel role for glucosamine as a desiccation stress-responsive metabolite in the underexplored tropical aquatic midge, Chironomus ramosus. Using high- throughput liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QToF-MS) analysis, biochemical assays and gene expression studies, we confirmed that glucosamine was essential during the recovery phase in C. ramosus larvae. Additionally, we demonstrated that trehalose, a known stress-protectant was crucial during desiccation but did not offer any advantage to the larvae during recovery. Based on our findings, we emphasise on the collaborative interplay of glucosamine and trehalose in conferring overall resilience to desiccation stress and propose the involvement of the trehalose-chitin metabolic interface in insects as one of the stress-management strategies to potentiate recovery post desiccation through recruitment of glucosamine.


Supplementary Figures
. Comparative view of the cross-sectional histology of the outer body integument of C. ramosus and D. melanogaster larva.
Histology-brief protocol: Bouin's fixed larvae were treated for serial dehydration in alcohols grades and paraffin embedding. Sections were deparaffinized in xylene and rehydrated in alcohol grades. Hematoxylin-eosin stained sections were examined under bright field microscope. presence of trehalose in larvae fed with trehalose prior to desiccation. Feeding was carried out for 1 h and larvae were sacrificed 5 min post feeding to confirm the uptake of trehalose.
We confirmed that the high peak signal is majorly due to exogenously fed trehalose (and to some extent due to basal endogenous trehalose levels) as compared to the corresponding low trehalose signal in the undesiccated control ( Fig. 2B          from trehalose. Glucosamine generated by trehalose hydrolysis is utilized for the synthesis of glucosamine 6-phosphate following a series of steps. Glucosamine 6-phosphate is then converted to N-acetyl glucosamine 6-P. Alternately, N-acetyl glucosamine 6-P can also be generated from glucosamine. After few more steps in the pathway, finally, polymerization of N-acetyl glucosamine produces chitin by CHS. Chitin can be degraded either by chitinase that releases free N-acetyl glucosamine monomers or by CDA which yields free glucosamine. The free glucosamine is amenable to the recycling process from where it can be again incorporated into the pathway.

Movie S1
Time-lapsed video demonstrating the desiccation and revival of a single larva of C.
ramosus. The first 30 s of the video shows actively moving larva in water (undesiccated control ). Next, the larva was transferred to Whatman filter paper to remove excess moisture adhering to the body. The larva was then gently picked up using a non-abrasive brush and transferred to a Petri dish lined with dry tissue paper and kept in the desiccator.
10 μl of water was given to the larva in order to prevent drying during the process of placing the larva inside the desiccator. Larva was then allowed to dehydrate inside the desiccator. Threshold desiccation tolerance of C. ramosus was 50±10 min. In this video, the larva was desiccated for 40 min. Thereafter, the desiccated larva was gently transferred using a brush and allowed to rehydrate in water. During the first 15-20 min of rehydration, larval body 'jerks' were observed followed by progressive signs of revival (increase frequency of wriggling) over the next 2h of rehydration min with active body movements.
Initial slight body movements characterized by 'jerks' and 'wriggling' were considered as the early signs of revival. Larval survival was judged by gentle stimulation with a brush.
With progressive rehydration, larval movements became more prominent and were now characterised by increased frequency of wriggling movements defined as 'undulatory movement' which was used as a behavioural parameter for judging larval recovery after desiccation stress. One larval undulation was considered as one complete wave-like motion, starting from the head and traversing in an antero-posterior direction which ends at the tip of the posterior parapods. We defined the beginning of this undulatory movement as larval recovery (details furnished in materials and methods).