Henry H. Hagedorn

Juvenile hormone and juvenile hormone esterase in adult females of the mosquito Aedes aegypti

Abstract Juvenile hormone III levels and juvenile hormone esterase activity were measured in whole body extracts and haemolymph, respectively, of female Aedes aegypti. The amount of juvenile hormone, determined by coupled gas chromatography-mass spectrometry, rose over the first 2 days after emergence from 0.7 to 7.5 ng/g, and then slowly fell over the next 5 days in females not given a blood meal. In females fed blood, juvenile hormone levels fell during the first 3 h to 2.3 ng/g. The rate of decline then slowed so that levels had reached their lowest point (0.4 ng/g) by 24 h after the blood meal. By 48 h, levels started to rise again until 96 h when they were equivalent to pre-blood meal levels. Juvenile hormone esterase activity in the haemolymph of females was measured with a partition assay. The esterase activity showed small fluctuations in unfed animals. In females fed blood on the 3rd day after emergence, the juvenile hormone esterase activity rose slowly to a peak at 36 h. At 42 h it began to decline, and by 66 h it had returned to pre-blood meal levels. Thus, juvenile hormone levels and juvenile hormone esterase activity were inversely correlated after a blood meal. Both the ovary and fat body produce juvenile hormone esterase in organ culture. Juvenile hormone III acid was the only metabolite produced after incubation of haemolymph with racemic-labelled juvenile hormone III. Juvenile hormone acid, diol, and acid diol were the main metabolic products seen in whole animal extracts after topical application of labelled hormone. About 25% of topically applied, labelled juvenile hormone appears in the haemolymph as the acid diol, and 50% of this is excreted in the urine immediately after the blood meal. Topical application of BEPAT (S-benzyl-O-ethyl phosphoramidothiolate), a specific inhibitor of juvenile hormone esterase, resulted in the absence of juvenile hormone acid and a reduction in the acid diol. Both BEPAT and methoprene, a juvenile hormone analogue, caused a reduction in egg hatch when applied topically 30 h after a blood meal, demonstrating that the decline in juvenile hormone levels after a blood meal is necessary for normal egg development and suggesting that the decline is mediated, at least in part, by juvenile hormone esterase.

Dynamic changes in flow rate and composition of urine during the post-bloodmeal diuresis inAedes aegypti (L.)

Summary1.The urine of blood-fed mosquitos was collected and analyzed for elemental composition and osmolality.2.Peak rates of urine flow averaged 4.9 droplets/min at 6 min following the beginning of the bloodmeal; peak flow urine contained, in mM, Na 178, K 4, and Cl 132, and the urine was approximately isosmotic to hemolymph.3.As urine flow rates fell, the [Na] of the urine decreased and the [K] increased. Urine osmolality declined, measuring less than 100 mOsm/kg in some samples, as compared to 354 mOsm/kg measured in pre-bloodmeal hemolymph.4.When urine flow rates had fallen to less than 10% of peak flow rates, urine again became approximately isosmotic to hemolymph, still containing Na and K as the principal cations.5.Approximately 40% each of the water, Na and Cl contained in the plasma fraction of the bloodmeal was excreted during the 1–2h period following the bloodmeal. This excretion represents only 20–30% of the total loads ingested in the bloodmeal.6.The data are consistent with dynamic changes in the functioning of both the Malpighian tubules and hindgut during the post-bloodmeal diuresis.

Haemolymph ecdysteroid in the housefly, Musca domestica, during oögenesis and its relationship with vitellogenin levels

Abstract Ecdysteroid titre in the haemolymph of the housefly, Musca domestica , cycled during oogenesis and peaked at ∼50 pg/μl during stages 5, 6 and 7. Levels of 10–20 pg/μl were found in houseflies with pre- and post-vitellogenic ovaries. Removal of the corpus allatum and corpus cardiacum complex resulted in low ecdysteroid levels (10 pg/μl). Ovariectomized flies also had lower ecdysteroid levels than the controls at 2 days (5 pg/μl) after emergence but not at 6 days (22 pg/μl). It is possible that the ecdysteroid peak that occurred during stages 5, 6 and 7 was produced by the ovaries because ovaries secreted and synthesized ecdysteroid in vitro . Endogenous haemolymph ecdysteroid levels had a linear correlation with the amount of vitellogenin that held for hormone concentrations of 5–43 pg/μl. Furthermore, the injection of 20-hydroxyecdysone at doses of 10 ng−1.0 μg/fly increased the amount of vitellogenin from 6 h to 12 h after injection; by 24 h, the vitellogenin returned to control levels. When 20-hydroxyecdysone was injected into ovariectomized flies, it was rapidly degraded and 96% was cleared from the haemolymph within 1 h.

Culture of honeybee organs: development of a new medium and the importance of tracheation

SummaryA culture system for honeybeen fat body and ovary was developed that supported optimal levels of protein synthesis by the explanted tissues. Abdominal body wall preparations of honeybee workers and queens, with adhering fat body, and ovaries of egg-laying queens were incubated in a culture medium designed to match honeybee hemolymph composition as closely as possible. Incorporation of [3H]eucine into soluble tissue proteins was measured. The new medium makes possible rates of tracer incorporation into fat body proteins that are up to three times higher than other media tested. When the tracheal system of the organs was let intact and open to the air during incubation, protein synthesis increased 17-fold (fat body) or 15-fold (ovary) as compared to preparations without open tracheas. After explantation into the medium, labeled proteins were synthesized at a highly variable rate for 10 h, probably due to wound response, and at a constant rate for the next 60 h. In contrast, ovarian protein synthesis occurred at a constant rate for at least 20 h and showed no wound response. The rate of tracer incorporation into fat body proteins was 3.2 times greater in tissues from the queen. This culture system is therefore suitable for a variety of investigations in honeybeen development and reproduction.

Isolation of mosquito vitellogenin genes and induction of expression by 20-hydroxyecdysone

Abstract Vitellogenic female Aedes aegypti contain abundant, 6500 nucleotide long RNAs that are not present in males or non-vitellogenic females and which were presumed to encode vitellogenin (VG). Three clones that hybridized to cDNA made to poly(A + )RNA from vitelogenic females, but not to cDNA made to male RNA, were selected from a genomic library. DNA from each clone hybridized to the 6500 nucleotide RNA species. Restriction enzyme mapping suggests the clones represent three distinct genes. The two that have been characterized share an uninterrupted region of homology about 6.5 kb long. Part of the coding region of one of the cloned genes was inserted into an expression vector, and the resulting polypeptide reacted specifically with antibodies to vitellogenin, thus confirming that the clones contain VG genes. Using one of the cloned genes as a probe on northern hybridizations we found that injection of 20-hydroxyecdysone into non-blood-fed decapitated females stimulated vitellogenin gene expression. The response was much greater in blood-fed decapitated females than in non-blood-fed females.

The role of factors from the head in the regulation of egg development in the mosquito Aedes aegypti

The relationships between the release of factors from the head after blood-feeding, subsequent levels of ecdysteroids and vitellin, and the ultimate maturation of eggs in Aedes aegypti were investigated. Females were decapitated at various times after a blood meal, at 20 or 48 h after feeding the animals were dissected and divided into two groups, those with arrested oocytes (yolk length 100 μm). These yolk lengths correspond with the levels of oocyte growth believed to accompany the proposed initiation and promotion phases of egg development. Animals dissected at 20 h were assayed for ecdysteroid by radioimmunoassay; those dissected at 48 h were assayed for vitellin by rocket immunoelectrophoresis. Non-blood-fed unoperated females contained 8% as much ecdysteroid as blood-fed controls and no measurable vitellin. Females with arrested oocytes (< 100 μm) were obtained only if decapitations were performed before 8 h; these females had about 20% of the ecdysteroids and 8% of the vitellogenin normally found in blood-fed animals. Females decapitated between 2 and 8 h with maturing oocytes contained 50–60% as much ecdysteroid and vitellin as blood-fed unoperated controls. Normal ecdysteroid and vitellin levels were reached only when decapitations were delayed for 12 and 24 h, respectively. The number of developing oocytes was also decreased by early decapitation and was closely correlated with vitellin levels. We conclude that the egg development neurosecretory hormone is released twice, once before 8 h and once after 8 h, to control ecdysteroid levels. We also suggest the presence of other factors from the head that control vitellin levels, the number of developing oocytes, and the early growth of the oocyte (initiation).

Development of responsiveness to hormones after a blood meal in the mosquito Aedes aegypti

Abstract The effects of exogenous hormones on oocyte development in isolated abdomens from blood-fed female Aedes aegypti were examined. Abdomens were prepared immediately after a blood meal. Single applications of hormones were administered immediately after ligation or 18 hr after the blood meal. Double applications were done at both times. Oocyte development was assayed by measuring the amount of yolk in oocytes 66 hr after the blood meal. Topical application of maximum doses of methoprene immediately after ligation caused oocytes to mature in 60% of the abdomens; a half-maximum response was obtained with 300 pg. Injection of 700 ng of 20-hydroxyecdysone (20-HE) was necessary to cause an equivalent response. Delaying the injection of 20-HE until 18 hr after feeding reduced the amount necessary to obtain a half-maximum response to 150 ng. Treating the abdomens twice dramatically reduced the amount of 20-HE needed for the second dose: pretreatment of abdomens immediately after ligation with 50 pg of 20-HE reduced the amount of 20-HE needed in the second injection to 30 ng. Pretreatment with a topical application of 50 pg of methoprene had a similar effect. These data indicate that the sensitivity of the mosquito to exogenous hormones changes after a blood meal, and that either 20-HE or methoprene can promote a further increase in sensitivity.

The vitellogenin gene family of Aedes aegypti

We have been interested in identifying genes that play a role in reproduction of the mosquito Aedes aegypti. Our interests are currently focused on the vitellogenin genes which in the mosquito are expressed only in the fat body in response to the insect steroid hormone, 20-hydroxyecdysone. Four of the five vitellogenin genes in the genome have been cloned. We have examined the relationships between these genes and find that they form a small gene family exhibiting different levels of relationship.

Ecdysteroid levels and integument changes in post-embryonic stages of Anastrepha suspensa

Ecdysteroid levels in larvae and pupae of Anastrepha suspensa (Diptera: Tephritidae) were measured by radioimmunoassay. These levels were correlated with histological changes throughout the development of the post-embryonic stages. Ecdysteroid levels increase rapidly throughout the last-larval instar and on the last day of this stage are 283 pg equivalents of 20-hydroxyecdysone per insect (14.5 ng/g) when wandering behaviour is initiated. At the end of this period the puparium is formed and within 24 h, the ecdysteroid rises to its highest peak (625 pg equivalents of 20-hydroxyecdysone/insect). Larval-pupal apolysis is initiated within 24 h later and the pupal cuticle is then secreted. Two days later, the ecdysteroids reach their lowest levels (75 pg equivalents of 20-hydroxyecdysone/insect or 0.6 ng/g) and most of the larval fat body and other tissues have been histolysed. In 5- to 10-day old pupae ecdysteroid levels again increase and remain relatively high throughout. During this period the larval epidermis is replaced by imaginal epidermis, imaginal discs begin to proliferate and the adult cuticle is secreted. Ecdysteroid levels finally fall 2 days prior to adult emergence. HPLC determinations indicate that 20-hydroxyecdysone is the predominant free ecdysteroid, and along with ecdysone, is readily detectable in all postembryonic stages of this species. An unusually high and unexplained peak of 20-hydroxyecdysone occurs in the pharate adult. This peak probably consists of ecdysone metabolites with retentions similar to that of 20-hydroxyecdysone and to which the antiserum is sensitive.