Effect of starvation on lipophorin density in fifth instar larval Manduca sexta.

Lipophorin (Lp) is a major insect lipoprotein and is responsible for lipid transport between organs. In this study, the effect of starvation on Lp properties was analyzed in larval Manduca sexta during the fifth instar. Lp hemolymph concentrations in larvae at days 1 and 2 were around 2-3 mg/ml and at day 3 it increased to 8 mg/ml. When larvae were starved for 24 h, they did not grow, but their body mass and hemolymph volume did not decrease significantly. Differences in Lp densities were observed. In fed larvae, from days 1 to 4, two major Lp populations were found with densities of 1.124 ± 0.002 (high density Lp-larval1 , HDLp-L1 ) and 1.141 ± 0.002 g/ml (HDLp-L2 ). When larvae were starved for 24 h, only one Lp population was present, with density 1.114 ± 0.001 g/ml (HDLp-Ls ). When larvae were abdominally ligated at day 1 or 2 of fifth instar, only HDLp-Ls was found after 24 h, indicating that the formation of this HDLp population was not dependent on any factor released by head. On the other hand, larvae that were ligated at day 3 showed the same Lp populations as the fed ones. In 24-h starved larvae, lipid load in Lp was higher as compared to the fed controls. In 24-h ligated larvae Lp lipid content increased when ligation was performed on day 1 or 2, but not on day 3. So, different responses to starvation can be observed depending on the developmental phase of the same larval instar.

Protease gene duplication and proteolytic activity in Drosophila female reproductive tracts.

Secreted proteases play integral roles in sexual reproduction in a broad range of taxa. In the genetic model Drosophila melanogaster, these molecules are thought to process peptides and activate enzymes inside female reproductive tracts, mediating critical postmating responses. A recent study of female reproductive tract proteins in the cactophilic fruit fly Drosophila arizonae, identified pervasive, lineage-specific gene duplication amongst secreted proteases. Here, we compare the evolutionary dynamics, biochemical nature, and physiological significance of secreted female reproductive serine endoproteases between D. arizonae and its congener D. melanogaster. We show that D. arizonae lower female reproductive tract (LFRT) proteins are significantly enriched for recently duplicated secreted proteases, particularly serine endoproteases, relative to D. melanogaster. Isolated lumen from D. arizonae LFRTs, furthermore, exhibits significant trypsin-like and elastase-like serine endoprotease activity, whereas no such activity is seen in D. melanogaster. Finally, trypsin- and elastase-like activity in D. arizonae female reproductive tracts is negatively regulated by mating. We propose that the intense proteolytic environment of the D. arizonae female reproductive tract relates to the extraordinary reproductive physiology of this species and that ongoing gene duplication amongst these proteases is an evolutionary consequence of sexual conflict.

TOR signaling is required for amino acid stimulation of early trypsin protein synthesis in the midgut of Aedes aegypti mosquitoes.

Blood meal digestion in mosquitoes occurs in two phases, an early phase that is translationally regulated, and a late phase that is transcriptionally regulated. Early trypsin is a well-characterized serine endoprotease that is representative of other early phase proteases in the midgut that are only synthesized after feeding. Since the kinase Target of Rapamycin (TOR) has been implicated as a nutrient sensor in other systems, including the mosquito fat body, we tested if TOR signaling is involved in early trypsin protein synthesis in the mosquito midgut in response to feeding. We found that ingestion of an amino acid meal by female mosquitoes induces early trypsin protein synthesis, coincident with phosphorylation of two known TOR target proteins, p70S6 kinase (S6K) and the translational repressor 4E-Binding Protein (4E-BP). Moreover, in vitro culturing of midguts from unfed mosquitoes led to amino acid-dependent phosphorylation of S6K and 4E-BP which could be blocked by treatment with rapamycin, a TOR-specific inhibitor. Lastly, by injecting mosquitoes with TOR double stranded RNA (dsRNA) or rapamycin, we demonstrated that TOR signaling was required in vivo for both phosphorylation of S6K and 4E-BP in the midgut, and for translation of early trypsin mRNA in response to amino acid feeding. It may be possible to target the TOR signaling pathway in the midgut to inhibit blood meal digestion, and thereby, decrease fecundity and the spread of mosquito borne diseases.

Utilization of pre-existing energy stores of female Aedes aegypti mosquitoes during the first gonotrophic cycle.

Pre-existing energy reserves may play an important role in regulating the utilization of blood meal proteins in female anautogenous mosquitoes. Determining the fate of reserves derived from the sugar meal and larval food during the first gonotrophic cycle would help to elucidate the relative contributions of larval and adult nutrition to survival and reproduction. We measured the allocation of pre-blood-meal reserves to egg production or energy production during the first gonotrophic cycle by using [14C]-labeled female Aedes aegypti mosquitoes. Feeding adults [3,4-14C]-glucose labeled the glycogen and sugar stores (approximately 50%), lipid stores (approximately 25%), and protein and amino acid stores (approximately 25%). During the first gonotrophic cycle, about 60% of the glycogen and sugar stores were metabolized and all were used for energy production. About 33% of the labeled protein and 72% of the labeled amino acid stores were metabolized, with about 9% being transferred to the eggs and the rest oxidized. About 30% of the lipid was metabolized, with about 65% being transferred to the eggs and the rest oxidized. Feeding [1-14C]-oleic acid to larvae effectively labeled adult lipid stores with about 75% of the label in lipid stores and 16% in proteins and 6% in glycogen. During the first gonotrophic cycle, about 35% of the labeled lipid stores were metabolized, with equal amounts being oxidized and transferred to the eggs. None of the other maternal stores labeled by fatty acid were metabolized during the first gonotrophic cycle. These results show that carbohydrate reserves are a critical source of energy during the first gonotrophic cycle, while lipid reserves are used equally for energy production and provisioning the eggs.

Triacylglycerol-rich lipophorins are found in the dipteran infraorder Culicomorpha, not just in mosquitoes.

Lipophorin is the major hemolymph protein responsible for lipid transport between tissues of insects. Lipophorins from several insect species in order Diptera (the fruit fly Drosophila melanogaster from the suborder Brachycera, the mosquito Aedes aegypti; the phantom midges Chaoborus maximus and minimus; the black fly Simulium vittatum; the crane fly Nephrotoma abbreviata, all from the suborder Nematocera) were isolated and characterized. All lipophorins consisted of two protein subunits of approximately 240 and 75 kDa each. The density of each lipophorin was in the high-density lipoprotein range (1.112 to 1.128 g/ml). The predominant neutral lipid carried by lipophorin from insects belonging to the infraorder Culicomorpha was triacylglycerol. Lipophorin from the crane fly Nephrotoma abbreviata, which belongs to the infraorder Tipulomorpha, carried approximately equivalent amounts of diacylglycerol and triacylglycerol. Lipophorin from D. melanogaster was found to carry diacylglycerol as the predominant neutral lipid.

A double blind, randomized, placebo-controlled study of SP-303 (Provir) in the symptomatic treatment of acute diarrhea among travelers to Jamaica and Mexico.

OBJECTIVE: The study was designed to evaluate the effectiveness of SP-303 (Provir), a plant-derived product with novel antisecretory properties, in the treatment of travelers' diarrhea. METHODS: A total of 184 persons from the United States who acquired diarrhea in Jamaica or Mexico were enrolled in a double-blind, placebo-controlled study examining the effectiveness of three doses of SP-303 in reducing illness. Subjects were treated with 125 mg, 250 mg, or 500 mg SP-303 or a matching placebo four times a day for 2 days. Subjects kept daily diaries of symptoms and were seen each day for 3 days. Of the subjects, 169 (92%) were included in the efficacy analysis. RESULTS: The most common etiological agent identified was enterotoxigenic Escherichia coli, found in 19% of subjects. The mean time interval from taking the first dose of medication until passage of the last unformed stool during 48 h therapy (TLUS48) was 38.7 h for the placebo group. TLUS48 was shortened by SP-303: 30.6 h for the 125-mg dose group (p = 0.005); 30.3 h for the 250-mg group; and 32.6 h for the 500-mg group (p = 0.01). Treatment failures were seen in 29.3% in the placebo group compared with 7.3% (p = 0.01), 4.3 (p = 0.002), and 9.8 (p = 0.026) in the three treatment groups. SP-303 was well tolerated at all doses. CONCLUSIONS: SP-303 was effective in shortening the duration of travelers' diarrhea by 21%. This antisecretory approach works directly against the pathophysiology of travelers' diarrhea and is not likely to potentiate invasive forms of diarrhea or to produce posttreatment constipation.