Luis A. Quesada-Allué

>Morphogenesis and cuticular markers during the larval-pupal transformation of the medfly Ceratitis capitata

Changes in morphology during early metamorphosis of the medfly, Ceratitis capitata (Wied.) (Tephritidae) were correlated with biochemical differentiation events. Protein profiles were studied both in the 3rd instar larval cuticle further transformed into puparium and the newly synthesized pupal cuticle. Beta‐alanine incorporation into the puparium (0–20 h) correlates with concomitant pigmentation (completed by 16 h) and sclerotization phenomena. This early ‘tannification program seems to be followed by deposition of a layer of substances, probably ecdysial fluid remnants, into the puparium. Their deposition ends approximately at +46 h. Simultaneously, pupal cuticle material starts to be deposited. Synthesis and deposition of the main pupal cuticle protein was detected 48 h after pupariation. At that time, eversion of the pupal head occurs. The definitive profile of pupal cuticle proteins was attained at around +72 h together with the establishment of adult body proportions.

The inhibition of insect chitin synthesis by tunicamycin

Summary The conversion of [14C]-labeled glucosamine to [14C]-labeled insect chitin was inhibited by tunicamycin, a nucleoside antibiotic which specifically blocks the biosynthesis of dolichyl diphosphate N-Acetylglucosamine. A method of short-term culturing is described for the analysis of chitin and glyconjugates biosynthesis by epidermal tissue of Triatoma infestans .

Metamorphosis-associated proteolysis in Ceratitis capitata

The induction of several biochemical indicators of larval tissue histolysis in the Medfly, Ceratitis capitata (Diptera: Tephritidae) (Wiedemann) was studied. Using synchronized third instar larvae, we have determined the time of occurence of gut evacuation (12 h before puparium formation, bpf), disappearance of digestive enzymes (10 h bpf), and jumping from the food (8 h bpf). We can also correlate these events temporally with other early landmarks of metamorphosis.

In vivo biosynthesis of a stage-specific cuticle glycoprotein during early metamorphosis of the medfly Ceratitis capitata

Cuticle proteins of an insect pest, the Medfly Ceratitis capitata, were resolved in polyacrylamide gels and partially characterized. The pupal cuticle was found to be different from cuticles of other insects since more than 80% w/w of the protein is a single mannose-containing polypeptide (PCG-100). The temporally-regulated in vivo biosynthesis and deposition of cuticle proteins was studied by microinjection of [35S]methionine followed by hand dissection of pupal cuticles. The major pupal glycoprotein, PCG-100, is cuticle- and stage-specific and was the earliest to be labeled and deposited. Its synthesis was maximal at around 46 hours after pupariation and then it decreased. The deposited PCG-100 and other minor pupal cuticle proteins become non-extractable at the end of the instar (7 days after pupariation) probably by sclerotization phenomena. These results provide insight into the temporal control of gene expression programs involved in cuticle deposition during medfly metamorphosis.

Analysis of survival, gene expression and behavior following chill-coma in the medfly Ceratitis capitata: Effects of population heterogeneity and age

The medfly Ceratitis capitata is an agricultural pest distributed worldwide thanks, in part, to its phenotypic plasticity of thermal tolerance. Cold exposure has been shown to reduce C. capitata survival, which may affect its distribution in areas with subfreezing temperatures. When insects are increasingly cooled, they attain a critical thermal threshold and enter a chill-coma state characterized by cessation of movement. It is not clear how a rapid cold exposure affects the physiological state of medflies, and how this is influenced by age and population heterogeneity. In order to approach these questions, C. capitata single-sex laboratory populations of 15 and 30 days old were subjected to a chill-coma recovery assay, and separated according to their recovery time in three subgroups: Fast-Subgroups, Intermediate-Subgroups, and Slow-Subgroups. Thereafter, we analyzed their survival, behavioral, and gene expression outputs. In female and old male populations, we found that flies with the slowest recovery time had a reduced life expectancy, a higher initial mortality rate, and a worse climbing performance compared with flies that recovered faster. Therefore, we were able to separate subgroups that developed chilling-injury from subgroups that had a reversible full recovery after cold exposure. The gene expression analysis of the heat shock protein genes hsp70 and hsp83 showed no clear association with the parameters studied. Interestingly, thorax expression levels of the Cu/Zn superoxide dismutase gene were elevated during the recovery phase in the Fast-Subgroups, but remained constant in the Slow-Subgroups that developed chilling-injury. On the other hand, none of the young male subgroups seemed to have suffered irreversible damage. Thus, we concluded that depending on age and population heterogeneity, chill-coma recovery time points out significant differences on individual cold tolerance. Moreover, the inability to properly induce the antioxidant defense system to counteract the oxidative damage caused by cold seems to contribute to the development of chilling-injury.

Chill-coma recovery time, age and sex determine lipid profiles in Ceratitis capitata tissues

The remodeling of membrane composition by changes in phospholipid head groups and fatty acids (FA) degree of unsaturation has been associated with the maintenance of membrane homeostasis under stress conditions. Overall lipid levels and the composition of cuticle lipids also influence insect stress resistance and tissue protection. In a previous study, we demonstrated differences in survival, behavior and Cu/Zn superoxide dismutase gene expression between subgroups of Ceratitis capitata flies that had a reversible recovery from chill-coma and those that developed chilling-injury. Here, we analyzed lipid profiles from comparable subgroups of 15 and 30-day-old flies separated according to their recovery time after a chill-coma treatment. Neutral and polar lipid classes of chill-coma subgroups were separated by thin layer chromatography and quantified by densitometry. FA composition of polar lipids of chill-coma subgroups and non-stressed flies was evaluated using gas chromatography coupled to mass spectrometry. Higher amounts of neutral lipids such as triglycerides, diacylglycerol, wax esters, sterol esters and free esters were found in male flies that recovered faster from chill-coma compared to slower flies. A multivariate analysis revealed changes in patterns of storage and cuticle lipids among subgroups both in males and females. FA unsaturation increased after cold exposure, and was higher in thorax of slower subgroups compared to faster subgroups. The changes in neutral lipid patterns and FA composition depended on recovery time, sex, age and body-part, and were not specifically associated with the development of chilling-injury. An analysis of phospholipid classes showed that the phosphatidylcholine to lysophosphatidylcholine ratio (PC/LPC) was significantly higher, or showed a tendency, in subgroups that may have developed chilling-injury compared to those with a reversible recovery from coma.

Metamorphosis and Gonad Maturation in the Horn Fly Haematobia irritans

Abstract The bloodsucking horn fly, Haematobia irritans (L.) (Diptera: Muscidae), is one of the most damaging pests of pasture cattle in many areas of the world. Both male and female imagoes spend their adult stage on the host, while immature stages develop in dung. Our goal was to determine if the progress of H. irritans gonad maturation can be correlated with eye and cuticle pigmentation events that occur during development of the imago within the puparium. The progression of germline cell divisions in immature gonads was analyzed from the beginning of the third larval instar (48 hours after egg hatch) until imago ecdysis. In the developing male larval gonad, meiosis began 72 hours after egg hatch, whereas in females oogonia were premeiotic at 72 hours. Meiosis was not detected in females until the mid-pharate adult stage, 120 hours after puparium formation. Therefore, gonad maturation in females appears to be delayed 144 hours with respect to that in males. In the stages within the puparium, the timing of germline cell division events was correlated with the progress of pigmentation of the eyes and cuticle as external markers.