Differential peptidomics highlights adipokinetic hormone as key player in regulating digestion in anautogenous flesh fly, Sarcophaga crassipalpis

Abstract

Females of anautogenous flesh flies, Sarcophaga crassipalpis, need a protein meal in order to produce their first batch of eggs. This protein meal elicits an increase in midgut proteolytic activity that is under neuropeptidergic regulation. Time series of decapitation and rescue experiments of liver fed flies evidenced the need of a peptide factor released by corpora cardiaca (CC) within four hours post protein feeding in order to assure complete protein digestion. Q-Exactive quantitative differential peptidomics analysis on CC of sugar fed flies and flies five hours post protein feeding respectively, showed a unique consistent decrease in the stored amount of adipokinetic hormone (AKH) ranging between 16 up to 63%. Injection of AKH into liver fed decapitated flies as well as sugar fed intact flies resulted in dose dependent enhanced midgut proteolytic activity up to the level of intact protein fed flies. This suggests a key role of AKH in food depended reproduction According to the reviewers minor remarks and suggestions and in response to your directions we made the appropriate changes as explained below:For Reviewer 1 the manuscript was acceptable as such and no action needed to be taken.As reviewer 2 felt uncomfortable with some of our figures as it was hard to understand each figure due to complicated representation on different food sources and time-courses, we adopted the graphical figures by incorporating line type explanation directly on the figures. In all experimental figures ( fig 3 and beyond) the broken line represent the non decapitated liver fed control situation whereas the dotted line represent the liver primed decapitated control condition and the solid line represent the liver primed decapitated experimental condition. According to directions we saved each figure file in .tiff format.As in Line 336-337 reviewer 2 asked to explain "a non-protein free amino acids" for readers. We replaced the sentence by "a free amino acids meal devoid of any protein" in line 337.3. As reviewer 2 informed about what kind of unit that was employed for enzymatic activity we adopted each figure as the relative enzymatic activity is expressed in absorption units as measured at 405 nm and normalized against the mass of the sample. Accordingly we explicitly clarified this in line 128 of M&M. We included (A 405nm ) in the figures ordinate We thank the reviewers for their suggestions and sincerely hope that the proposed changes suffice to make this manuscript ready for publication. Kind regards Prof.Dr.Roger Huybrechts Cover LetterResponse to reviews According to the reviewers minor remarks and suggestions and in response to your directions we made the appropriate changes as explained below:For Reviewer 1 the manuscript was acceptable as such and no action needed to be taken.As reviewer 2 felt uncomfortable with some of our figures as it was hard to understand each figure due to complicated representation on different food sources and time-courses, we adopted the graphical figures by incorporating line type e...

Characterization of the adipokinetic hormone receptor of the anautogenous flesh fly, Sarcophaga crassipalpis

“…As expected, this also led to an increase of the feeding frequency of that insect (Konuma et al, 2012). Furthermore, post liver feeding decrease of the AKHR gene expression level corresponds well with the decrease of the AKH peptide observed in liver fed flesh flies (Bil et al, 2014).…”

“…Based on differences in quantity of AKH peptide observed between sugar and liver fed female flies (Bil et al, 2014), the receptor transcript tissue distribution analysis was also performed on sugar and protein fed (5 h post protein feeding) flies. Significantly reduced AKHR transcript levels were measured in the brain, fat body, hindgut and midgut (p-values T-student test: p brain = 0.020; p fat body = 0.018; p hindgut = 0.018; p midgut = 0.009) of liver fed females (Fig.…”

“…For this reason, the control of feeding and digestion processes is crucial for the anautogenous reproductive strategy. One of the neuropeptides involved in the midgut enzymatic activity regulation of the flesh fly Sarcophaga crassipalpis is adipokinetic hormone (AKH) (Bil et al, 2014).…”

Characterization and pharmacological analysis of two adipokinetic hormone receptor variants of the tsetse fly, Glossina morsitans morsitans

“…A similar stomatogastric nervous system is present in the tsetse fly (Langley, 1965), where AKH neuropeptides may be released from axon termini and regulate feeding, digestion or nutrient transport. Previous reports indicated a hormonal role for insect AKH in the regulation of midgut, salivary gland, and crop functioning (Bil et al, 2014;Kodrík et al, 2012;Vinokurov et al, 2014;Stoffolano et al, 2014), but it remains elusive whether neuronal projections innervating these tissues or local production of AKH is involved.…”

PHARMACOLOGICAL REGULATION OF DIGESTION IN THE ANAUTOGENOUS FLESH FLY, Sarcophaga crassipalpis, BY SIMPLE INJECTION OF 6‐HYDROXYDOPAMINE

“…Cages with flies selected for experiments were sugar and water diet only to ensure no earlier development of ovaries. The other cages with flies that were not used were fed with a piece of cow liver to continue normal eggs development and larviposition (Bil et al., ).…”