Physiological characterization and regulation of the contractile properties of the mosquito ventral diverticulum (crop)

Abstract

In adult dipteran insects (flies), the crop is a diverticulum of the esophagus that serves as a food storage organ. The crop pumps stored contents into the alimentary canal for digestion and absorption. The pumping is mediated by peristaltic contractions of the crop musculature. In adult female mosquitoes, the crop (ventral diverticulum) selectively stores sugar solutions (e.g., nectar); proteinaceous blood meals by-pass the crop and are transferred directly to the midgut for digestion. The mechanisms that regulate crop contractions have never been investigated in mosquitoes. Here we provide the first physiological characterization of the contractile properties of the mosquito crop and explore the mechanisms that regulate crop contractions. Using an in vitro bioassay we found that the isolated crop spontaneously contracts in Ringer solution for at least 1 hour and its contractions are dependent on extracellular Ca2+. Adding serotonin (5- hydroxytryptamine, 5-HT) or a membrane-permeable analog of cyclic adenosine monophosphate (cAMP) to the extracellular bath increased the frequency of crop contractions. On the other hand, adding benzethonium chloride (BzCl; a chemical that mimics the effects of myosuppressins), H-89 or Rp-cAMPS (inhibitors of protein kinase A, PKA), or carbenoxolone (an inhibitor of gap junctions) reduced the frequency of the unstimulated, spontaneous and/or 5-HT-stimulated crop contractions. Adding aedeskinin III did not detectably alter crop contraction rates. In addition to pharmacological evidence of gap junctions, we demonstrated that the crop expressed several mRNAs encoding gap junctional proteins (i.e. innexins). Furthermore, we localized immunoreactivity for innexin 2 and innexin 3 to muscle and epithelial cells of the crop, respectively. Our results 1) suggest that 5-HT and myosupressins oppositely regulate contractile activity of the mosquito crop, and 2) provide the first evidence for putative roles of cAMP, PKA, and gap junctions in modulating contractile activity of the dipteran crop.

Stop the crop: insights into the insecticidal mode of action of cinnamodial against mosquitoes

“…These contraction frequencies were averaged together and referred to as the 'treatment' rate. Our previous study (Calkins et al, 2017) and preliminary trials in the present study found that the solvents did not affect contraction rates at a 1% final assay concentration. In some experiments a 'pre-treatment' period with CDIAL, 5-HT, or octopamine (OA) preceded the 'treatment' period.…”

“…We demonstrated previously that the crop of Ae. aegypti undergoes spontaneous peristaltic contractions in vitro that are Ca 2+ -dependent, stimulated by 5-hydroxytryptamine (5-HT), and inhibited by a chemical mimic of myosupressins, benzethonium chloride (Calkins et al, 2017). Here we demonstrate that CDIAL completely inhibits the spontaneous and 5-HT stimulated contractions of the crop in vitro.…”

“…Crops were isolated from non-blood fed adult females (3-10 days post-emergence) under mosquito Ringer's solution as previously described (Calkins et al, 2017). If a crop was leaking its contents, filled with air bubbles, or not contracting spontaneously, then it was discarded.…”

“…We utilized an in vitro assay previously established for mosquito crops (Calkins et al, 2017). In brief, an isolated crop or hindgut was transferred to a single well of a 96-well microtiter plate (USA Scientific, Ocala, FL) containing 100 µl of Ringer's solution where it rested for 5 min.…”

Fly foregut and transmission of microbes

“…Because the adult crop in mosquitoes has been ignored, very few physiological studies have focused on mechanisms of crop regulation. Recently, Calkins et al (2017) examined some of these in adult female A. aegypti and found the factors involved in the modulation of crop contractions are very similar to those previously reported in other dipterans (Stoffolano and Haselton, 2013). Being such an important vector of microbial pathogens to humans, these studies on the crop and microbes of mosquitoes opens up a new avenue of research that may ultimately provide a mechanism(s), in addition to the attractive toxic sugar bait (ATSB) technique (Wongthangsiri et al, 2018), that is providing a new control strategy for insect pests.…”