Effect of Selection for Pyrethroid Resistance on Abiotic Stress Tolerance in Aedes aegypti from Merida, Yucatan, Mexico.

The study of fitness costs of insecticide resistance mutations in Aedes aegypti has generally been focused on life history parameters such as fecundity, mortality, and energy reserves. In this study we sought to investigate whether trade-offs might also exist between insecticide resistance and other abiotic stress resistance parameters. We evaluated the effects of the selection for permethrin resistance specifically on larval salinity and thermal tolerance. A population of A. aegypti originally from Southern Mexico was split into two strains, one selected for permethrin resistance and the other not. Larvae were reared at different salinities, and the fourth instar larvae were subjected to acute thermal stress; then, survival to both stresses was compared between strains. Contrary to our predictions, we found that insecticide resistance correlated with significantly enhanced larval thermotolerance. We found no clear difference in salinity tolerance between strains. This result suggests that insecticide resistance does not necessarily carry trade-offs in all traits affecting fitness and that successful insecticide resistance management strategies must account for genetic associations between insecticide resistance and abiotic stress resistance, as well as traditional life history parameters.

Aerobic function in mitochondria persists beyond death by heat stress in insects.

The critical thermal maximum (CTmax) of insects can be determined using flow-through thermolimit respirometry. It has been demonstrated that respiratory patterns cease and insects do not recover once the CTmax temperature has been reached. However, if high temperatures are maintained following the CTmax, researchers have observed a curious phenomenon whereby the insect body releases a large burst of carbon dioxide at a rate and magnitude that often exceed that of the live insect. This carbon dioxide release has been termed the post-mortal peak (PMP). We demonstrate here that the PMP is observed only at high temperatures, is oxygen-dependent, is prevented by cyanide exposure, and is associated with concomitant consumption of oxygen. We conclude that the PMP derives from highly active, aerobic metabolism in the mitochondria. The insect tracheal system contains air-filled tubes that reach deep into the tissues and allow mitochondria access to oxygen even upon organismal death. This unique condition permits the investigation of mitochondrial function during thermal failure in a manner that cannot be achieved using vertebrate organisms or in vitro preparations.

Thermal acclimation in a complex life cycle: the effects of larval and adult thermal conditions on metabolic rate and heat resistance in Culex pipiens (Diptera: Culicidae).

It has now been well established that insects can respond to variation in their environment via acclimation, yet the extent of the response varies among populations and environmental characteristics. One under-investigated theme which may contribute to this variation concerns acclimation effects across the life cycle. The present study explores how acclimation in the larval stage of Culex pipiens affects thermal relations in the adult stage. Mosquitoes were reared in a full factorial design at 18 or 26 °C as larvae and adults, then critical thermal maxima (CTmax) and metabolic rate-temperature relationships (MR-T) were determined for all 4 treatments. CTmax was positively affected by both larval and adult acclimation treatments. MR-T slope was significantly affected only by adult treatment: warm acclimated adults had on average shallower slopes and higher y-intercepts than cool acclimated ones. These results demonstrate that larval acclimation effects can alter adult phenotypes in a species whose life cycle includes two drastically different environments, an aquatic and a terrestrial stage. Studying insects with complex life cycles, especially those with aquatic or subterranean larval stages, can provide valuable information on the effects of thermal variability and predictability on phenotypic plasticity.

Thermal resistance and performance correlate with climate in populations of a widespread mosquito.

The abundance and success of widely distributed species across variable environments make them suitable models for exploring which traits will be important for resilience to climate change. Using a widespread mosquito species, Culex tarsalis, we have investigated population-level variation in the critical thermal maximum (CT(max)) and the metabolic response to temperature (MR-T). Adult female C. tarsalis were sampled from three sites representing thermally distinct habitats in California, and flow-through respirometry was used to determine CT(max) and MR-T relationships. CT(max) differed significantly among the three populations and correlated positively with maximum temperatures at each site but not with mean temperatures. Culex tarsalis from our cool-temperature, high-altitude site had significantly higher metabolic rates at each test temperature compared with the two populations from warmer sites, consistent with previous examples of thermal compensation in ectothermic animals inhabiting cold climates. The MR-T slope was steepest in mosquitoes inhabiting the site with the lowest temperature variability, while shallower slopes were exhibited by mosquitoes from the two sites with higher thermal variability. Our results show the extent to which local populations may differentiate within their respective environments and suggest that plasticity in thermal tolerance traits may play a role in mediating resilience to climate change. Furthermore, our study highlights the importance of thermal variability and extremes rather than average temperatures for the evolution of thermal traits.

Inversion 2La is associated with enhanced desiccation resistance in Anopheles gambiae.

BACKGROUND: Anopheles gambiae, the principal vector of malignant malaria in Africa, occupies a wide range of habitats. Environmental flexibility may be conferred by a number of chromosomal inversions non-randomly associated with aridity, including 2La. The purpose of this study was to determine the physiological mechanisms associated with the 2La inversion that may result in the preferential survival of its carriers in hygrically-stressful environments. METHODS: Two homokaryotypic populations of A. gambiae (inverted 2La and standard 2L+(a)) were created from a parental laboratory colony polymorphic for 2La and standard for all other known inversions. Desiccation resistance, water, energy and dry mass of adult females of both populations were compared at several ages and following acclimation to a more arid environment. RESULTS: Females carrying 2La were significantly more resistant to desiccation than 2L+(a) females at emergence and four days post-emergence, for different reasons. Teneral 2La females had lower rates of water loss than their 2L+(a) counterparts, while at four days, 2La females had higher initial water content. No differences in desiccation resistance were found at eight days, with or without acclimation. However, acclimation resulted in both populations significantly reducing their rates of water loss and increasing their desiccation resistance. Acclimation had contrasting effects on the body characteristics of the two populations: 2La females boosted their glycogen stores and decreased lipids, whereas 2La females did the contrary. CONCLUSION: Variation in rates of water loss and response to acclimation are associated with alternative arrangements of the 2La inversion. Understanding the mechanisms underlying these traits will help explain how inversion polymorphisms permit exploitation of a heterogeneous environment by this disease vector.

2La chromosomal inversion enhances thermal tolerance of Anopheles gambiae larvae.

BACKGROUND: The mosquito Anopheles gambiae is broadly distributed throughout sub-Saharan Africa and this contributes to making it the most efficient vector of malaria on the continent. The pervasiveness of this species is hypothesized to originate in local adaptations facilitated by inversion polymorphisms. One inversion, named 2La, is strongly associated with aridity clines in West and Central Africa: while 2La is fixed in arid savannas, the 2L+a arrangement is predominantly found in the rainforest. Ability to survive high temperature exposure is an essential component of aridity tolerance, particularly in immature stages that are restricted to shallow puddles. Toward deciphering the role of the 2La inversion in local adaptation, the present investigation focused on variation in larval and pupal thermo-tolerance in two populations dissimilar solely in 2La arrangement. METHODS: A laboratory colony of A. gambiae that is polymorphic for 2La but standard for all other known inversions was used to create 2 homokaryotypic populations (2L+a and 2La). The survival of 4th instar larvae and pupae from both populations was then tested following exposure to thermal stress with and without prior heat hardening. RESULTS: Larvae responded identically to a 40 degrees C heat stress, with about 50% of larvae dying after 1.5-2 h and few larvae surviving a 3 h stress. When heat hardened prior to the thermal stress, thermo-tolerance of both larval populations increased, with 2La 24 h survival significantly exceeding that of 2L+a. Pupae were generally more thermo-tolerant than larvae, although 2La pupae were less so than 2L+a. Heat hardening had no positive effect on pupal thermo-tolerance. CONCLUSION: The increased thermo-tolerance observed in 2La larvae following heat hardening suggests higher responsiveness (i.e., thermal sensitivity) of the inverted karyotype. By responding more drastically to the heat shock, 2La larvae are better equipped to resist the potentially lethal temperatures that occur in arid habitats. The lower survival of 2La pupae compared with 2L+a may reflect the cost of this sensitivity, whereby the thermal resistance mechanisms prevent successful completion of metamorphosis. The costs and benefits of thermal resistance are discussed in light of the climates characterizing either end of the 2La frequency cline.

Bias, precision and accuracy in the estimation of cuticular and respiratory water loss: a case study from a highly variable cockroach, Perisphaeria sp.

We compared the precision, bias and accuracy of two techniques that were recently proposed to estimate the contributions of cuticular and respiratory water loss to total water loss in insects. We performed measurements of VCO2 and VH2O in normoxia, hyperoxia and anoxia using flow through respirometry on single individuals of the highly variable cockroach Perisphaeria sp. to compare estimates of cuticular and respiratory water loss (CWL and RWL) obtained by the VH2O-VCO2 y-intercept method with those obtained by the hyperoxic switch method. Precision was determined by assessing the repeatability of values obtained whereas bias was assessed by comparing the methods' results to each other and to values for other species found in the literature. We found that CWL was highly repeatable by both methods (R0.88) and resulted in similar values to measures of CWL determined during the closed-phase of discontinuous gas exchange (DGE). Repeatability of RWL was much lower (R=0.40) and significant only in the case of the hyperoxic method. RWL derived from the hyperoxic method is higher (by 0.044 micromol min(-1)) than that obtained from the method traditionally used for measuring water loss during the closed-phase of DGE, suggesting that in the past RWL may have been underestimated. The very low cuticular permeability of this species (3.88 microg cm(-2) h(-1) Torr(-1)) is reasonable given the seasonally hot and dry habitat where it lives. We also tested the hygric hypothesis proposed to account for the evolution of discontinuous gas exchange cycles and found no effect of respiratory pattern on RWL, although the ratio of mean VH2O to VCO2 was higher for continuous patterns compared with discontinuous ones.

Evidence from mosquitoes suggests that cyclic gas exchange and discontinuous gas exchange are two manifestations of a single respiratory pattern.

In this paper we demonstrate that the apparent pattern of gas exchange in insects, as observed using flow-through respirometry, is strongly affected by the rate of flow of air through the system. This is true not only because of the time constant of the respiratory chamber in which the insect resides, but also due to the effect of flow rate on the residence time of air as it passes through the detection chamber in the gas analyzer. It is demonstrated that insects respiring with a discontinuous gas exchange pattern can appear to be using a cyclic respiratory pattern. The effects of flow rate on the respiratory pattern discerned are illustrated using the mosquito Culiseta inornata. It is demonstrated that these mosquitoes respire discontinuously. They are among the smallest insects to date in which the discontinuous gas exchange cycle has been observed.

Physiology of desiccation resistance in Anopheles gambiae and Anopheles arabiensis.

Desiccation resistance and water balance were studied in the adult female mosquitoes Anopheles arabiensis and Anopheles gambiae sensu stricto. When the two species were reared from egg to adult under identical conditions, An. arabiensis had significantly higher desiccation resistance than did An. gambiae. Data are presented that indicate that this difference in desiccation resistance is associated with a higher body water content prior to desiccation in An. arabiensis. No differences in rate of water loss during desiccation or water content at death were observed. Measurements of metabolic rate and respiratory pattern also showed no statistically significant differences between the species. This study provides the first physiologic measurements of desiccation resistance in adults of these species and offers insights into the physiologic differences associated with differential resistance to desiccation stress.

Metabolic rate in female Culex tarsalis (Diptera: Culicidae): age, size, activity, and feeding effects.

We used flow-through respirometry on female mosquitoes to observe individual ventilatory pattern and to measure metabolic rate at rest, during activity and after a blood-meal. At rest, young adult females of the species Culex tarsalis ventilated cyclically with an average VCO2 of 6.5 nl/min and frequency of 45 mHz. Both resting metabolic rate and body mass of females increased between emergence and 4 d, but only body mass continued to increase until 12 d. Mating status had no effect on the body mass or resting metabolic rate of the females. Both lipid and carbohydrate stores strongly increased during the first week of adult life, in particular between emergence and 4 d, in which lipid mass increased 8.3-fold and carbohydrate mass increased 3.6-fold. Hourly metabolic rate after a blood or sugar meal was measured over a 6-d period in two groups of 14 females. Sugar-fed mosquitoes remained calm during the day and had nocturnal increases in metabolic rate associated with flight activity. Blood feeding elicited a specific dynamic action lasting approximately 55 h, at the peak of which metabolic rate of the blood-fed females was twice that of the sugar-fed group. Eggs were laid on the third night. The increase in metabolic rate presumably reflected the cost of blood digestion and egg production. The females were not active during digestion, so that although their metabolic rate was increased, the overall energy expenditure of the blood-fed group was not very different from that of the sugar-fed group.