Hemolymph circulation in insect sensory appendages: functional mechanics of antennal accessory pulsatile organs (auxiliary hearts) in the mosquitoAnopheles gambiae

Abstract

Mosquito antennae provide sensory input that modulates hostseeking, mating and oviposition behaviors. Thus, mosquitoes must ensure the efficient transport of molecules into and out of these appendages. To accomplish this, mosquitoes and other insects have evolved antennal accessory pulsatile organs (APOs) that drive hemolymph into the antennal space. This study characterizes the structural mechanics of hemolymph propulsion throughout the antennae of Anopheles gambiae. Using intravital video imaging, we show that mosquitoes possess paired antennal APOs that are located on each side of the head's dorsal midline. They are situated between the frons and the vertex in an area that is dorsal to the antenna but ventral to the medial-most region of the compound eyes. Antennal APOs contract in synchrony at 1 Hz, which is 45% slower than the heart. By means of histology and intravital imaging, we show that each antennal APO propels hemolymph into the antenna through an antennal vessel that traverses the length of the appendage and has an effective diameter of 1-2 μm. When hemolymph reaches the end of the appendage, it is discharged into the antennal hemocoel and returns to the head. Because a narrow vessel empties into a larger cavity, hemolymph travels up the antenna at 0.2 mm s −1 but reduces its velocity by 75% as it returns to the head. Finally, treatment of mosquitoes with the anesthetic agent FlyNap (triethylamine) increases both antennal APO and heart contraction rates. In summary, this study presents a comprehensive functional characterization of circulatory physiology in the mosquito antennae.KEY WORDS: Antenna, Hemocoel, Circulatory physiology, Antennal heart, Heart, Vessel INTRODUCTIONThe antennae of insects are involved in the detection of chemical, tactile, thermal and auditory stimuli (Chapman and Simpson, 2013). The antennae also mechanically assist during the mating of fleas, and contain the timing elements of the sun compass that drives the autumn migration of monarch butterflies (Hsu and Wu, 2001;Merlin et al., 2009). In mosquitoes, the detection of near-field sound by the antennae's Johnston's organs influences courtship and mating, and the reception of odorant and thermal cues by antennal receptor neurons controls oviposition and host-seeking behaviors (Cator et al., 2009;Wang et al., 2009;Carey et al., 2010;Rinker et al., 2013). Because antennae are intricately involved in essential physiological processes, insects must ensure that molecules required for proper RESEARCH ARTICLEDepartment of Biological Sciences, Vanderbilt University, Nashville, TN 37235, USA. antennal functioning are efficiently transported into and out of these peripheral sensory appendages.The primary manner by which insects transport nutrients, hormones, waste and other molecules from one region of the body to another is by circulating a fluid medium called hemolymph through an open body cavity called the hemocoel Klowden, 2013). The main pump that drives hemolymph circulation is called the dorsal vessel, which is a muscula...

CCAP and FMRFamide-like peptides accelerate the contraction rate of the antennal accessory pulsatile organs (auxiliary hearts) of mosquitoes

“…The general procedure used to inject and visualize the contractions of the antennal APOs and the heart was essentially as we have described previously (Boppana and Hillyer, 2014). Briefly, under a Nikon SMZ645 stereomicroscope (Nikon, Tokyo, Japan), a mosquito was injected with ∼0.2 µl of 0.1% solids 1 μm diameter neutral density yellow-green (505/515) carboxylate-modified fluorescent microspheres (Molecular Probes, Eugene, OR, USA) in either phosphate buffered saline (PBS; pH 7.0; used as the vehicle solution) or in PBS containing CCAP (H-PFCNAFTGC-NH 2 , including a disulfide bond between the cysteines), FMRFamide (H-FMRF-NH 2 ) or SALDKNFMRFamide (H-SALDKNFMRF-NH 2 ) at a concentration of 1×10 −6 mol l −1 (all peptides were purchased from Bachem Americas, Torrance, CA, USA).…”

“…Contractions of each antennal APO were counted manually by visualizing the contraction-driven movement of the fluorescent microspheres that had aggregated at each APO (Boppana and Hillyer, 2014). Contractions of the heart were counted manually by annotating each wave-like contraction of cardiac muscle that shifted the fluorescent microspheres that had aggregated at the periostial regions of the heart (King and Hillyer, 2012;Boppana and Hillyer, 2014).…”

“…After hemolymph reaches the distal end of this appendage, it flows back to the head via the antennal hemocoel (Clements, 1956;Sun and Schmidt, 1997;Boppana and Hillyer, 2014). Intravital imaging experiments that visualized the flow of hemolymph confirmed these findings, and demonstrated that hemolymph is propelled to the distal end of the antennae at a velocity that is four times faster than the velocity in which it flows back toward the head (Boppana and Hillyer, 2014).…”

“…In addition to the dorsal vessel, insects employ accessory pulsatile organs (APOs), also called auxiliary hearts, to propel hemolymph into narrow areas of the body, or areas that are distant from the dorsal vessel (Pass, 2000;Pass et al, 2006). Depending on the species, these APOs are located at the base of the antennae, wings and ovipositor, and in the ventral abdomen (Pass, 1991;Richter and Hertel, 1997;Matus and Pass, 1999;Andereck et al, 2010;Boppana and Hillyer, 2014;Hustert et al, 2014;Wipfler and Pass, 2014;Pass et al, 2015).…”

“…After hemolymph reaches the distal end of this appendage, it flows back to the head via the antennal hemocoel (Clements, 1956;Sun and Schmidt, 1997;Boppana and Hillyer, 2014). Intravital imaging experiments that visualized the flow of hemolymph confirmed these findings, and demonstrated that hemolymph is propelled to the distal end of the antennae at a velocity that is four times faster than the velocity in which it flows back toward the head (Boppana and Hillyer, 2014). Although no investigations have tested the effect of endogenous factors on the contraction rate of the antennal APOs of mosquitoes, the neuropeptides CCAP, FMRFamide and SALDKNFMRFamide, and the neurotransmitters serotonin and glutamate, have been shown to accelerate the heart rate of these insects (Estevez-Lao et al, 2013;Hillyer et al, 2014Hillyer et al, , 2015.…”