Mosquitoes transmit a diverse range of pathogens6–10, and thus, an improved understanding of mosquito physiology and the development of novel control strategies are critically important. AL has afforded us new insights into mosquito larval respiration. The application of AL resulted in the expulsion of gas bubbles that originated from within the tracheal system. When the impinging acoustic frequency is equal to the resonant frequency of the gas within the tracheal system, that gas maximally absorbs acoustic energy and begins to pulsate in synchrony with the impinging frequency. As energy continues to be applied, the amplitude of the pulsation increases to the point of rupturing the DTTs. By adjusting the amplitude and pulse length of our acoustic signal, we observed the earliest manifestations resulting from AL, the severing of the DTTs, with minimal collateral tissue trauma. This Figure 4. Tracheal gases blocked from passing through the Tracheal Occlusion (TO). Note the gas (yellow arrow) internal to the DTTs is differentiated from liquid by higher transparency and refraction of light showing spectrum colors. This indicates the TO restricts the movement of gas between the tracheal system and external environment. The PLs are in a partial open condition. Ae. aegypti perispircular lobes (PL), Felt Chamber (FC).
Figure 5. Survival of Ae. aegypti larvae (plotted using Kaplan Meier survival curve) (n = 23) with ruptured DTTs after exposure to sublethal acoustic energy. Some mosquitoes died within 24 hours of acoustic exposure, others survived for an extended period, with visible damage to their DTTs. Note, four treated mosquitoes pupated; these mosquitoes were removed from further observation and are shown on the graph as pupation events. novel technique allowed us to reveal several new aspects of the larval tracheal system. Our study has five major outcomes. First, we improved our understanding of the mosquito tracheal system, including the possible isolation of the tracheal system from the atmosphere. Second, we presented a potential mechanism for the maintenance of pressure during impingement, which damages the DTTs (as opposed to gas venting through the siphon). Third, we provided new insights into the morphology of the siphon (the identification of the TO). Fourth, we confirmed that the damage-inducing mechanism of action of AL is the acoustic resonance of the gas within the tracheal system. Finally, we observed that the siphon does not play an obligate role in respiration for the following reasons:
The TO appears to isolate and maintain the tracheal system at an elevated pressure thus making it at best an inefficient port for the two-way exchange of metabolic gasses. Larvae with completely blocked spiracles or severed DTTs continued to live for long periods of time. After acoustic exposure we did not observe any hemolymph (liquid, solids or gasses) pass by the TO as would have been expected if the siphon was open to the atmosphere.
Margaret L. Keister reported “… a survey of the literature (see Wigglesworth,’31) shows that there are numerous gaps and contradictions in our knowledge of insect tracheal systems”11. Our anatomical findings complement a recent revival of interest in mosquito respiratory physiology. However, some conflicts still exist 12–16.
Accordingly, it is important to define some terms used herein. The “FC” is identified by Keilin, Tate and Vincent as the terminal chamber between the spiracles and DTT17. An appreciation of the physiology of the DTTs is important in analyzing gas volumes. Regarding “active DTTs”, during the development of a given instar between molts, the DTTs are comprised of a chitinous partially gas-filled trunk; the active DTTs are enclosed in the large and fluid-filled living-tissue trunks of the future-instar DTTs (Supplemental 1a). However, the composition of this fluid is unknown18,19 and needs further investigation. The active DTTs are withdrawn during molting. While resting on the water surface with the five PLs extended, it appeared that mosquito larvae were in an ideal position to freely exchange metabolic gases, intake oxygen and expel carbon dioxide. Our results indicated that there was not an obligate need for this, and beyond the incidental cuticular exchange of gas with the atmosphere in the atrium, the direct exchange of tracheal gases with the atmosphere (breathing) is unlikely.
By comparing various anatomical and physical characteristics before and after acoustic exposure we identified the tracheal system to be at an elevated pressure. Mosquito larvae are nearly neutrally buoyant20. They are composed of solids, liquids and gasses. In order to maintain their buoyant condition, the volume proportions of the gas filled tracheal system to body volume must fall within a precise range. As reported by Ha, this percentage for Anopheles sinensis larvae was.34%16. The results of our observations and calculations using 37 A. aegypti samples was.33%. This is expected as most of the body is liquid therefore only a small percentage of body volume could be gas. We calculated that the mean post acoustic exposure proportion of gas bubbles to body volume is 2.02%.
This represents and expansion of 5.9 times meaning the initial pressure in the tracheal system was high. The mean direct expansion of the gas bubble was 5.0 times that of as original tracheal size. The function of pressurization in the DTT is unknown, and its potential relationship with tracheal filling or emergence should be further studied.
A pressurized tracheal system makes the inhalation of oxygen difficult if not impossible. The TO is quite strong because it involves acoustically induced pressure oscillations that exceed the ability of the DTTs to contain them. The dimensions of the TO between surface-resting or submerged larvae do not change, suggesting that the restriction prohibits the exhaust of carbon dioxide. Dissection to sever the siphon anterior to the FCs also rendered AL ineffective, indicating that the TO is a necessary structure for the success of AL.
The condition of total dependence on cuticular (and/or gill- or filament-supported) respiration in immature aquatic insects is common and present in many close relatives of mosquitoes. Culicinae are joined by seven other families in the infraorder Culicomorpha whose immatures all respire in total submergence 21. The consideration that the siphon plays only a vestigial role in respiration is not without precedent. Corethra (also called midge and of the family Chaoboridae) were classified as mosquitoes until the early 1960s22; today, they are considered taxonomically separate but are thought to share a common ancestor 21. Corethra and mosquito larvae share common physiological traits, and many species look very much alike, including the presence of an apparent larval siphon 21–26. Krogh found that larvae of the genus Corethra appeared to respire through only the skin and concluded that this organism fills its air sacks with gas from a non-atmospheric source (i.e., tissues)27. He also noted that the DTTs did not contain air and that the connected bladders appeared to have no respiratory function.
Mochlonyx spp. (also in Chaoboridae) possess a siphon (Supplemental 3) but do not come to the surface to breathe 25,26,28,29; hence, the siphon is clearly not used for the exchange of atmospheric gases in this species. As noted by Förster and Woods, and Keister and Buck, filling of the tracheal system with gasses from an endogenous source has been observed in a variety of organisms 11,18,19.
It has been previously reported that mosquito larvae can survive for long periods of time without access to the atmosphere, indicating that aquatic respiration is possible30–36. For example, Macfie (1917) demonstrated that submerged larvae (isolated from surface air) of certain mosquito species can live for 20 days if the aquatic medium is adequately aerated. Mosquito larvae are found in aquatic environments with variable levels of dissolved oxygen32,37–40 and can survive in water with low levels of dissolved oxygen concentrations (e.g., 0.04 to 1.63 mg/L). However, we propose that previous reports on mosquito survivability in low dissolved oxygen concentrations failed to consider that the dissolved oxygen content at the surface of water is higher from that further down in the water column. Vacha and others noted that the concentration of dissolved oxygen at the air-water interface was enhanced41,42.
Mosquito larvae in a surface-resting posture position their body, especially their ventral fan, in the stratum with the highest oxygen concentration while simultaneously conserving energy. This may be an important behavior in sourcing metabolic oxygen. Therefore, future investigations should focus on the role of the ventral fan in mosquito larval respiration. Our observations may question the commonly accepted mode of action (suffocation) of petroleum surfactants. According to the literature, the most rapid mode of action may be neurological disruption, not suffocation30. Suffocation normally takes a long time, which may be related to reduced surface oxygen concentrations or the direct impairment of cutaneous gas exchange43–49.
Our results show that the tracheal system is isolated and maintained at an elevated pressure, thus making the free exchange of metabolic gases with the environment unlikely. We report a previously undescribed TO that appeared to isolate the tracheal system and enabled acoustic energy to intensify and rupture the DTT. Our findings are not without precedence; as with other family members in the infraorder Culicomorpha, it is common for immatures to totally source metabolic oxygen from and expel waste gases directly into the water. These findings appear to contradict the fundamental understanding of culicine larval respiration. Additional tests and research evaluating gas movements through the environment as well as within the animals needs to be done. With the new physical and physiological information from this study, possibly novel methods to control this deadly insect will be developed.
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