Thomas M. Clark

Evolution and adaptive significance of larval midgut alkalinization in the insect superorder Mecopterida

The phylogenetic distribution of reported midgut pH values among larval Mecopterida supports a model in which the common ancestor of this group possessed an alkaline midgut, with subsequent loss of this trait in the lineage leading to the muscomorphan Diptera. The relationship between midgut pH and diet guild rank within the Lepidoptera and Diptera was tested by assigning numerical values to diet guilds (i.e., fruit, grasses, herbs, trees and shrubs, and organic detritus). Lepidopteran superfamilies were found to differ significantly in both midgut pH and in diet guild rank. Regression of mean superfamily midgut pH against mean superfamily diet guild rank yielded an R2 of 0.79 (N = 10), whereas regression of species midgut pH against species diet guild rank yielded an R2 of only 0.15 (N = 60). Species feeding on foliage of plant taxa high in tannins and on Solanaceae have midgut pH values above 9, and midgut pH in species feeding on these taxa is positively related to diet guild. In contrast, species feeding on the foliage of plant taxa containing terpenes, DIMBOA, glucosinolates, and pyrrolizidine alkaloids have midgut pH values near 8, and midgut pH of these species is either not related to diet guild (all species) or is negatively related to diet guild rank when the analysis is limited to the Noctuoidea. The data suggest that decreased midgut pH in species feeding on plants containing terpenes, DIMBOA, glucosinolates, and pyrrolizidine alkaloids may be an adaptive response that overrides selection for high pH in the presence of tannins and that midgut pH may be one factor contributing to the limitation of the host plant range of many species of lepidopteran herbivores.

Differences in the effects of salinity on larval growth and developmental programs of a freshwater and a euryhaline mosquito species (Insecta: Diptera, Culicidae)

SUMMARY The effects of salinity on growth and development of the euryhaline Ochlerotatus taeniorhynchus and the freshwater Aedes aegypti are compared. O. taeniorhynchus grow larger, and have greater intrinsic growth rates, than A. aegypti. Females of each species attain greater mass, take longer to develop, and have greater growth rates than do males. In O. taeniorhynchus, pupal mass, larval stage duration and growth rates (dry mass) increase with salinity, whereas growth rates (wet mass) remain constant across salinities, reflecting a decrease in percent body water. The pupal mass (wet or dry) of O. taeniorhynchus is determined primarily by effects of salinity on the rate of assimilation of dry mass, because the latter contributes very strongly to final pupal mass in both species. In contrast, the duration of A. aegypti larval stage follows a υ-shaped curve, with most rapid development at intermediate salinities. Growth rates of A. aegypti decrease with increasing salinity, and percent body water is constant across salinities. As for O. taeniorhynchus, duration of A. aegypti larval stage increases at high salinity. However, this increase in larval stage duration cannot compensate for the decrease in growth rate at high salinity, resulting in an overall decrease in both wet and dry pupal mass at high salinity. Thus, salinity has fundamentally different effects on developmental programs and phenotypic plasticity in the two species investigated.

Stimulation of Malpighian tubules from larval Aedes aegypti by secretagogues

Dibutyryl-cAMP (db-cAMP) and 5-hydroxytryptamine (5-HT) stimulate secretion rates of fluid, sodium, and potassium by Malpighian tubules from larval female Aedes aegypti, without affecting the sodium and potassium concentrations of the secreted fluid. High-potassium saline extracts of larval and adult tissues also cause increased fluid secretion rates by these tubules. The maximal rate of fluid secretion is agonist-dependent, and tubules secrete at the greatest rates in response to stimulation by 5-HT. Bumetanide has no effect on the fluid secretion rates of larval female tubules stimulated with 5-HT or db-cAMP. The head is the most concentrated source of diuretic neurosecretory material in larvae. The amount of material released into high-potassium saline from a single larval head causes a maximal response by a Malpighian tubule in vitro. The diuretic material in high-potassium saline extracts of larval heads is a peptide or peptides, apparently of small molecular weight. Enzymatic digestion studies reveal that it probably has a capped N-terminus, and a sialyl group is not required for activity. Size exclusion chromatography followed by reversed-phase HPLC yields a single peak of diuretic activity.

Dose-dependent effects of CRF-like diuretic peptide on transcellular and paracellular transport pathways

The mechanism of action of synthetic Culexcorticotropin-releasing factor (CRF)-like diuretic peptide (CCRF-DP) was investigated in isolated, perfused Malpighian tubules of the yellow fever mosquito, Aedes aegypti. Low concentrations of CCRF-DP (10-10 and 10-9 M) caused depolarizing oscillations of the lumen-positive transepithelial voltage ( V t) in Malpighian tubules, whereas high concentrations (10-8 and 10-7 M) first depolarized and then transiently hyperpolarized V t; CCRF-DP always lowered transepithelial resistance ( R t), regardless of voltage depolarization or hyperpolarization. The short-circuit current ( I sc), an electrical estimate of active transepithelial transport of Na and K, remained unchanged at low concentrations of CCRF-DP, but I sc more than doubled at high concentrations. These effects of CCRF-DP suggest dose-dependent sites of action: low concentrations of CCRF-DP affect the paracellular pathway, and high concentrations affect both paracellular and transcellular pathways.

pH tolerances and regulatory abilities of freshwater and euryhaline Aedine mosquito larvae

SUMMARY The pH regulatory abilities of two members of the mosquito tribe Aedini, known to have dramatically different saline tolerances, are investigated. The freshwater mosquito Aedes aegypti and the euryhaline Ochlerotatus taeniorhynchus tolerate very similar pH ranges. Both species complete larval development in waters ranging from pH 4 to pH 11, but naïve larvae always die in water of pH 3 or 12. Across the pH range 4–11, the hemolymph pH of O. taeniorhynchus is maintained constant while that of A. aegypti varies by 0.1 pH units. The salt composition of the water (3.5 g l–1 sea salt, 3.5 g l–1 NaCl, or nominally salt-free) has no effect on the range of pH tolerated by A. aegypti. In both species, the effects of pH on larval growth and development are minor in comparison with the influence of species and sex. Acclimation of A. aegypti to pH 4 or 11 increases survival times in pH 3 or 12, respectively, and allows a small percentage of larvae to pupate successfully at these extreme pH values. Such acclimation does not compromise survival at the other pH extreme.

Alkalinization by Manduca sexta anterior midgut in vitro: requirements and characteristics

Abstract Manduca sexta anterior midgut alkalinizes its luminal contents in vivo, achieving pH values up to 12 with rates of up to 81.4 μ Eq OH − h −1 . The alkalinization rate dropped to 3.0 μ Eq h −1 within minutes of mounting the tissue in an Ussing chamber, even though K + secretion (as measured by short-circuit transepithelial current and open circuit voltage) continued at a high rate for several hours. In contrast, cannulation of the anterior midgut in situ resulted in a preparation that alkalinized at rates up to 127 μ Eq h −1 for several hours. Removal of the head or ventral nerve cord did not interrupt alkalinization. However, alkalinization could be blocked by increasing the volume of the saline in which the basal side was bathed. The buffer capacity of the saline had no effect on the maximum pH attained or the rate of base secretion. Doubling of the drip rate from 0.5 to 1.0 ml min −1 had no effect on the maximum rate of OH − secretion but resulted in a reduction of the maximum pH attained from 11 to 8.5. These data suggest release of an alkalinization factor from the basal aspect of the gut that may act by altering the passive H + permeability of the tissue.

Strategies for regulation of hemolymph pH in acidic and alkaline water by the larval mosquito Aedes aegypti (L.) (Diptera; Culicidae).

SUMMARY The responses of larval Aedes aegypti to media of pH 4, 7 and 11 provide evidence for pH regulatory strategies. Drinking rates in pH 4 media were elevated 3- to 5-fold above those observed in pH 7 or 11. Total body water was elevated during acute exposure to acidic media. During chronic exposure, total body water was decreased and Malpighian tubule mitochondrial luminosity, quantified using Mitotracker Green FM, increased. Malpighian tubule secretion rates and energy demands thus appear to increase dramatically during acid exposure. In alkaline media, drinking rates were quite low. Larvae in pH 11 media excreted net acid (0.12 nequiv H+ g–1 h–1) and the pH indicators azolitmin and bromothymol blue revealed that the rectal lumen is acidic in vivo at all ambient pH values. The anal papillae (AP) were found to be highly permeant to acid–base equivalents. Ambient pH influenced the length, and the mass-specific length, of the AP in the presence of NaCl (59.9 mmol l–1). In contrast, the length and mass-specific length of AP were not influenced by ambient pH in low NaCl conditions. Mitochondrial luminosity was reduced in AP of larvae reared in acidic media, and was not elevated in alkaline media, relative to that of larvae reared in neutral media. These data suggest that the AP may compromise acid–base balance in acidic media, and may also be an important site of trade-offs between H+ homeostasis and NaCl uptake in dilute, acidic media.

Additive Effects of 5-HT and Diuretic Peptide on Aedes Malpighian Tubule Fluid Secretion

Abstract We have demonstrated previously that fluid secretion rates of Malpighian tubules from larval Aedes aegypti are stimulated by a diuretic peptide and by 5-hydroxytryptamine (5-HT, serotonin). In the present manuscript we demonstrate that diuretic peptide and 5-HT have additive effects on Malpighian tubule fluid secretion rates at low 5-HT concentrations (1–100 nM), while Malpighian tubules stimulated by higher 5-HT concentrations (1–10 μ M) do not respond to diuretic peptide in the fluid secretion bioassay. Fluid secretion rates are stimulated by forskolin and dibutyryl cAMP, but are unaffected by IBMX. Cyclic AMP concentrations are stimulated by 5-HT in vitro , while diuretic peptide has no effect on cAMP concentrations. These effects strongly support stimulation of adenylate cyclase activity by 5-HT and suggest that 5-HT and diuretic peptide act through separate second messenger systems.

Effects of serotonergic agents on survival and hemolymph composition of the larval mosquito Aedes aegypti (Diptera: Culicidae, L.) in vivo: does serotonin regulate hemolymph acid-base homeostasis?

SUMMARY The role of serotonin in the regulation of larval Aedes aegypti hemolymph composition was investigated in vivo using two reuptake inhibitors (SSRIs), alaproclate HCl and 6-nitroquipazine maleate, and the receptor antagonist methiothepin mesylate. Larvae were placed in media differing in pH and salinity in the presence and absence of serotonergic agents. The toxicity of each agent was strongly influenced by ambient pH. For each agent, toxicity was negligible in acidic media, intermediate in neutral media and greatest in alkaline media. By contrast, toxicity of all agents was independent of salinity. No effects on mass-specific body water or hemolymph volume were observed whereas hemolymph osmotic pressure, Na+ concentrations and pH differed significantly among treatments. 6-nitroquipazine caused a decrease in Na+ from 115±1.7 to 103±0.9 mmol l–1, and alaproclate caused alkalosis of the hemolymph from pH 7.55±0.026 to pH 7.72±0.044. Methiothepin decreased hemolymph osmotic pressure from 329±9.9 to 304±8.8 and showed the greatest overall toxicity. Control larvae excreted net base in pH 4 media (1.4 μmol g–1 h–1) and net acid in pH 7 (1.2 μmol g–1 h–1) and pH 11 (5.1μ mol g–1 h–1) media. In pH 4 media, alaproclate and methiothepin caused a shift to net H+ excretion (1.1 and 1.5 μmol g–1 h1, respectively) whereas these agents did not influence acid excretion rates in pH 7 or pH 11 media. The hypothesis that serotonin is involved in hemolymph acid–base balance is discussed.

Modulation of appetite and feeding behavior of the larval mosquito Aedes aegypti by the serotonin-selective reuptake inhibitor paroxetine: shifts between distinct feeding modes and the influence of feeding status

The effects of the serotonin-selective reuptake inhibitor paroxetine (2×10−5 mol l−1) on behavior of the larval mosquito Aedes aegypti are described. Four discrete behavioral states dominate larval behavior: wriggling, two distinct types of feeding, and quiescence. Feeding behaviors consist of foraging along the bottom of the container (substrate browsing), and stationary filter feeding while suspended from the surface film. Fed larvae respond to paroxetine with increased wriggling, and reductions in both feeding behaviors. In contrast, food-deprived larvae treated with paroxetine show no change in the proportion of time spent wriggling or feeding, but shift from stationary filter feeding to substrate browsing. Thus, actions of paroxetine in fed larvae are consistent with suppression of appetite and stimulation of wriggling, whereas paroxetine causes food-deprived larvae to switch from one feeding behavior to another. Further analysis of unfed larvae revealed that paroxetine decreased the power stroke frequency during wriggling locomotion, but had no effect on the swimming velocity during either wriggling or substrate browsing. These data suggest that: (1) serotonergic pathways may trigger shifts between distinct behaviors by actions on higher level (brain) integrating centers where behaviors such as feeding and locomotion are coordinated; (2) these centers in fed and food-deprived larvae respond differently to serotonergic stimulation suggesting sensory feedback from feeding status; and (3) serotonergic pathways also modulate central pattern generators of the nerve cord where the bursts of action potentials originate that drive the rhythmic muscle contractions of wriggling.