Methoprene treatment and its effect on male reproductive organ size and female remating in a fruit fly.

Methoprene, a juvenile hormone analog, is used to accelerate sexual maturation in males of species of economic importance in support to the sterile insect technique (SIT). In the SIT, mass-reared sterile males are released into the field and need to survive until they reach sexual maturation, find a wild female, mate with her and then induce female sexual refractoriness, so she will not remate with a wild counterpart. The use of methoprene shortens the time between release and copulation. However, in South American fruit flies, Anastrepha fraterculus, the ability of methoprene-treated males to inhibit female remating has been shown to be lower than wild males, when methoprene was applied by pupal immersion or topical application. Here we evaluated the possibility of incorporating methoprene into the male diet at different doses and the ability of those males to inhibit female remating, as well as the effect of methoprene on male reproductive organ size, due to the possible correlation between male accessory gland size and their content, and the role of male accessory gland proteins in female inhibition. We found that A. fraterculus males fed with methoprene in the adult protein diet at doses as high as 1% were less likely to inhibit female remating, however, at all other lower doses males had the same ability as untreated males to inhibit female remating. Males fed with methoprene had bigger male accessory glands and testes compared to methoprene-deprived males. We demonstrate that the incorporation of methoprene in adult male diets is possible in this species and potentially useful as a post-teneral, pre-release supplement at doses as low as 0.01%. Even at higher doses, the percentage of females remating after 48 h from the first copulation is sufficiently low in this species so as not compromise the efficiency of the SIT.

Post-mating gene expression of Mexican fruit fly females: disentangling the effects of the male accessory glands.

Mating has profound physiological and behavioural consequences for female insects. During copulation, female insects typically receive not only sperm, but a complex ejaculate containing hundreds of proteins and other molecules from male reproductive tissues, primarily the reproductive accessory glands. The post-mating phenotypes affected by male accessory gland (MAG) proteins include egg development, attraction to oviposition hosts, mating, attractiveness, sperm storage, feeding and lifespan. In the Mexican fruit fly, Anastrepha ludens, mating increases egg production and the latency to remating. However, previous studies have not found a clear relationship between injection of MAG products and oviposition or remating inhibition in this species. We used RNA-seq to study gene expression in mated, unmated and MAG-injected females to understand the potential mating- and MAG-regulated genes and pathways in A. ludens. Both mating and MAG-injection regulated transcripts and pathways related to egg development. Other transcripts regulated by mating included those with orthologs predicted to be involved in immune response, musculature and chemosensory perception, whereas those regulated by MAG-injection were predicted to be involved in translational control, sugar regulation, diet detoxification and lifespan determination. These results suggest new phenotypes that may be influenced by seminal fluid molecules in A. ludens. Understanding these influences is critical for developing novel tools to manage A. ludens.

Diagnosis of the new genus emAgnathia/em (Pycnogonida, Callipallenidae) and description of two new species from southern Australia.

A new genus of Callipallenidae, Agnathia, is established to accommodate two new species of pycnogonid from southern Australia; A. aria and A. chuki. Both new species are represented by adult and sub-adult forms. Gravid females and ovigerous and larvigerous males are represented. Postembryonic growth stages are recorded and briefly discussed. The presence of six-segmented ovigers in males of one species, as opposed to the usual ten segments in both sexes, is recorded. Genera that share morphological relationships are discussed and a key to these genera is provided. The genera Bradypallene, Pycnopallene and Safropallene are reassessed and reassigned to family incertae sedis.

The Sterile Insect Technique: Success and Perspectives in the Neotropics.

The sterile insect technique (SIT), an environmentally friendly means of control, is currently used against plant, animal, and human pests under the area-wide integrated pest management. It consists in the mass production, sterilization, and release of insects in an affected area where sterile males mate with wild females leading to no reproduction. Here, we review SIT in the Neotropics and focus on particular recent successful cases of eradication of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), as well as effective programs used against the Mexican fruit fly Anastrepha ludens (Loew), the New World screwworm fly Cochliomyia hominivorax (Coquerel)), and the Cactus moth Cactoblastis cactorum (Berg). We examine when SIT does not work and innovations that have made SIT more efficient and also highlight complimentary techniques that can be used in conjunction. We address potential candidate species that could be controlled through SIT, for example Philornis downsi Dodge & Aitken. Finally, we consider the impact of climate change in the context of the use of the SIT against these pests. Given the recent dramatic decline in insect biodiversity, investing in environmentally friendly means of pest control should be a priority. We conclude that SIT should be promoted in the region, and leadership and political will is needed for continued success of SIT in the Neotropics.

When Less Is More: Sex Ratios for the Mass-Rearing of Anastrepha ludens (Diptera: Tephritidae).

Anastrepha ludens (Loew) is one of the most important pests of citrus and mango crops in Mexico. A method used to control this pest is the sterile insect technique, which consists in the mass production, irradiation, and release of insects in affected areas. The production of insects begins with the establishment of colonies to produce eggs, which must be highly fertile to ensure an adequate production of larvae. However, female fecundity and fertility can be affected by adult density and sex ratio, thus an optimal sex ratio in mass-rearing cages must be used. The genetic sexing strain of A. ludens (Tapachula-7) allows the identification of the sex at the pupal stage, making it possible to establish rearing cages with different sex ratios. We determined if different sex ratios have an effect on egg production. Two sex ratios (4♀: 1♂ and 1♀: 1♂) were compared. Fecundity, fertility and survival at different ages were also determined. Higher fertility and fecundity per female were observed at a ratio of 4:1. However, females with higher fecundity had reduced survival probabilities. In conclusion, maintaining colonies with a lower proportion of males in cages ensures a greater fecundity and fertility. Further research is necessary to understand whether results can be attributed to lower male harassment in cages.

Identification of Ceratitis capitata (Diptera: Tephritidae) Sperm From Females in Traps: The Importance of the Ventral Receptacle.

The monitoring of a pest population often relies on the identification of individuals from traps. For area-wide programs utilizing the sterile insect technique, the further identification of the mated status of females found in traps is of utmost importance. For the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), this is usually verified through the assessment of sperm in the spermathecae. However, this can be misleading for species where there are other sperm storage organs such as the ventral receptacle. Here, we studied the relative importance of sperm storage in the ventral receptacle compared to the spermathecae for females from 5 to 18 d of age. Furthermore, we studied how sperm can be identified in the ventral receptacle or spermathecae after females were recovered from traps. We found no effect of female age on likelihood of sperm storage. Sperm could be identified in both sperm storage organs at 7 or 14 d after females had been placed in traps. We found that the ventral receptacle is a more reliable indicator of female mated status. Thus, we propose that if no sperm are found in the spermathecae, program managers should revise the ventral receptacle before assuming that females are not mated. This test may also be relevant to other pest tephritids that store even more sperm in the ventral receptacle than C. capitata.

Antipredator behavior of the new mass-reared unisexual strain of the Mexican Fruit Fly.

Tephritid pests controlled through the sterile insect technique (SIT) are mass-reared and subsequently released in affected areas. Several quality parameters are currently used to test adults, but none take into account interactions with a predator. When sterile males are released in the field, they will need to avoid predators until they reach sexual maturity and survive long enough to mate with wild females. Spiders are one of the most common predators that flies may encounter in release sites. In this study, we evaluated the antipredator behavior of a mass-reared sterile unisexual strain ('Tapachula-7') of the Mexican fruit fly Anastrepha ludens (Diptera: Tephritidae) against their spider predators. We sampled spiders in citrus trees to determine which families could be more common. We established the baseline activity rates of sterile Tapachula-7 (Tap-7) flies in comparison with wild flies. We also tested the behavior of the fertile and sterile bisexual strain and wild flies against hunting spiders (Family Salticidae) and orb building spiders (Family Tetragnathidae). We recorded 18 spider families, with Salticidae being the most dominant. Tap-7 flies diminished their activity in comparison with wild males at 1800 h but showed similar activity levels earlier in the day. When exposed to orb-web spiders (Leucauge venusta), Tap-7, fertile and sterile males from the bisexual strain had similar rates of survival, but Tap-7 males showed lower survival than wild males. Against hunting spiders (Phidippus audax), wild males had higher probability of defensive wing displays, but there was no difference in spider attack rates. In general, sterile Tap -7 males performed as well as males from the bisexual strain, although they had lower survival than wild males. This could be due to either mass-rearing and/or irradiation effects. We recommend the use of the defensive wing display behavior as a quality parameter and propose a rapid and effective method to evaluate fly activity. The efficiency of SIT will be improved if released sterile males have the same antipredator repertoire as their wild counterparts.

Effects of size, sex and teneral resources on the resistance to hydric stress in the tephritid fruit fly Anastrepha ludens.

Water availability is recognized as one of the most important factors in the distribution and activity of terrestrial organisms. In the case of insects, hydric stress imposes a major challenge for survival because of the small surface-area-to-volume ratio they exhibit. In general, stress resistance is expected to co-vary positively with size; however, this pattern can become obscured in insects that exhibit sexual size dimorphism, as sexes differ in size and/or shape and have dissimilar resource allocations. In the present study, we use an allometric-based approach to (i) assess the desiccation and starvation stress resistance of teneral Anastrepha ludens flies, (ii) disentangle the relationships between resistance, size and sex and (iii) examine the adult fly body differences in water and lipid contents before and after exposure to stress. After controlling for sexual size dimorphism, an allometric increase of resistance with overall size was observed for all stress-based treatments. The scaling exponents that define the proportion of increase resistance varied with size traits and with type and degree of hydric stress. In this allometric relationship, and also in the relationships between mass and wing length and between size and teneral resources, the sexes maintained similar scaling exponents but differed in the intercepts. Males were more resistant to stress than females; this competitive advantage is probably linked to greater amounts of teneral lipids and more water use during stress.

Male and female condition influence mating performance and sexual receptivity in two tropical fruit flies (Diptera: Tephritidae) with contrasting life histories.

Recent recognition of widespread polyandry in insects has generated considerable interest in understanding why females mate multiple times and in identifying factors that affect mating rate and inhibit female remating. However, little attention has been paid to understanding the question from both a female and male perspective, particularly with respect to factors that may simultaneously influence female remating rates. Here, we report on a study aimed at ascertaining the possible interactive effects that male and female size and diet, and female access to a host could have on mating latency, probability, and duration and female refractory period using two tropical fruit fly species with contrasting life histories. Of all factors tested, adult diet played the most significant role. Both Anastrepha ludens and Anastrepha obliqua males which had constant access to protein and sucrose mated more often, had shorter copulations and induced longer refractory periods in females than males fed a low quality diet (sucrose offered every third day). Female size and the interaction with male diet determined how quickly female A. ludens mated for the first time. Smaller females mated sooner with low quality fed males than with high quality fed males while there was no difference for large females, suggesting that male choice may be at play if high quality fed males discriminate against smaller females. Copulation duration also depended on both male and female nutritional condition, and the interaction between male diet and female size and diet. Large and high quality fed females had shorter copulations regardless of male condition. Importantly, for A. ludens, female refractory period depended on male size and the nutritional condition of both males and females, which could indicate that for this species, female receptivity does not depend only on the condition of the male ejaculate. For A. obliqua refractory period was associated with the interaction between male size and diet and male diet and host presence. We discuss our results in terms of male ability to inhibit female remating and the relative contribution of female condition to this behavior. We also address the importance of studying effects simultaneously on species with contrasting life histories.

Random mating among Anastrepha ludens (Diptera: Tephritidae) adults of geographically distant and ecologically distinct populations in Mexico.

The Mexican fruit fly Anastrepha ludens (Loew) (Diptera: Tephritidae) is a polyphagous pestiferous insect with a geographical range encompassing highly variable environmental conditions. Considering that cryptic species have been recently found among South American representatives of the same taxonomic group as A. ludens, we tested whether or not some populations of A. ludens have evolved assortative mating as an isolating mechanism that maintains intrapopulation genetic differences and behavioral adaptations to local conditions. Males and females stemming from widely separated locations with similar environmental conditions and males and females stemming from populations within individual-flight range, but collected in different hosts (a native and an exotic one), mated randomly amongst themselves when placed in a field cage. Despite the fact that sibling males and females from two distinct populations also mated randomly amongst themselves, siblings engaged in significantly longer copulations than non-siblings, indicating that perhaps adults discriminated mates with similar genetic compositions. Our results have important practical implications as A. ludens is the most devastating pest of citrus in Mexico and Central America, and large-scale releases of sterile flies are used to control it.

Identification of a C-terminal cdc25 sequence required for promotion of germinal vesicle breakdown.

Glutathione S-transferase (GST)-cdc25B(31-566) induced germinal vesicle breakdown (GVBD) when microinjected into Xenopus oocytes. Purified, N-terminally truncated forms of cdc25B did not induce GVBD, even though many had phosphatase activity and activated cdc2 in vitro. N-terminally truncated forms of cdc25B inhibited induction of GVBD by longer forms of the enzyme suggesting a direct interaction in vivo. cdc25B(356-556), but not cdc25B(364-529), inhibited GVBD induction by GST-cdc25B(31-566) suggesting that a region of cdc25B near to the C-terminus was responsible for the inhibition. To determine the region of peptide sequence that was inhibitory, cdc25B(356-556) was subjected to proteolysis with endoproteinase lys-C. Following a demonstration that the resulting peptide mixture inhibited GST-cdc25B-dependent GVBD, a series of peptides spanning amino acids at the C-terminus were synthesized. The peptide TRSWAGERSR inhibited GVBD induced by GST-cdc25B. An alanine scan of the peptide revealed residues critical for GVBD inhibition, and site-directed mutagenesis of the corresponding residues in GST-cdc25B(31-566) eliminated its ability to induce GVBD. These results demonstrate that a cdc25B C-terminal domain, involved in dominant-negative inhibition of GVBD-competent cdc25B, is required for induction of GVBD following microinjection into oocytes.

Rifapentine and isoniazid in the continuation phase of a 6-month regimen. Interim report: no activity of isoniazid in the continuation phase.

SETTING: Clinical trial amongst 762 patients with newly diagnosed pulmonary tuberculosis in Hong Kong. After an initial 2 months of a four-drug intensive phase consisting of streptomycin, isoniazid, rifampicin and pyrazinamide (SHRZ), a random allocation in continuation to once-weekly rifapentine + isoniazid (HRp1), HRp1 given in 2 of every 3 weeks (HRp1.2/3), or to three times weekly isoniazid + rifampicin (HR3). OBJECTIVE: Interim report evaluating progress of study and the role of isoniazid in the continuation phase. METHODS: Kaplan-Meier analysis and response of patients related to susceptibility of pretreatment organisms to isoniazid and to rate of isoniazid acetylation determined by NAT2 genotyping. RESULTS: In the 30-month follow-up, rates for adverse treatment events (failure and relapse) were 4.2% in the HR3, 10.2% in the HRp1 and 11.2% in the HRp1.2/3 series (P = 0.02 for HR3 vs HRp1 and P = 0.01 for HR3 vs HRp1.2/3). Occurrence of adverse events was not related to initial susceptibility to isoniazid nor to the rate of acetylation of isoniazid. CONCLUSIONS: The two rifapentine regimens had similar final rates of adverse events which were unsatisfactory. Isoniazid had little or no activity in the continuation phase, indicating that no improvement of the continuation regimen is likely to be obtained by alteration of the isoniazid dosage.

Comparative biological activities of alpha-MSH antagonists in vertebrate pigment cells.

We have previously reported that melatonin was an effective lightening agonist in the teleost Synbranchus marmoratus, the amphibians Rana pipiens and Bufo ictericus, and in the lizard Anolis carolinensis. The hormone, previously applied to the preparations, effectively inhibited alpha-MSH darkening activity in a dose-independent manner, and was also able to reverse MSH-induced darkening. We presently describe the inhibitory effect of the indoleamine on the murine melanoma cell proliferation. Interestingly, the hormone also stimulated tyrosinase activity, with a correlated increase in melanin content. We also demonstrate that in a diverse lizard species, Urosaurus ornatus, the indoleamine was totally ineffective. The competitive MSH antagonistic activity of H-His-D-Arg-Ala-Trp-D-Phe-Lys-NH2 has been demonstrated previously in R. pipiens and U. ornatus. Herein, its inhibitory activity is also reported in another lizard species, A. carolinensis. However, this MSH analogue was inactive in S. marmoratus, and in murine melanoma cells. On the other hand, the 7 thru 10 alpha-MSH fragment, Ac-Phe-Arg-Trp-Gly-NH2, although ineffective in S. marmoratus and R. pipiens, was an alpha-MSH antagonist in A. carolinensis. Surprisingly, in the melanoma cell line, the MSH fragment exhibited no agonist or antagonist activity, but dramatically potentiated the MSH-induced increase in tyrosinase activity. These data might suggest that the fragment is participating either in the process of facilitation or in positive cooperativity. The present results, taken together with our previously reported data, demonstrate a major interspecies diversity of the MC1 subtype of melanocortin receptor, and point out the relevance of the membrane microenvironment for the final receptor configuration.

A scintillation proximity assay for UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase.

A rapid and simple method for quantitating the reaction product of UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase (GalNAc-transferase) by scintillation proximity assay (SPA) was developed. The assay quantitates the radioactivity incorporated from 3H-labeled UDP-GalNAc into a biotin-labeled acceptor peptide, as measured after adsorption of the acceptor peptide to avidin-coated SPA beads. The acceptor peptide, PPASTSAPG (Elhammer et al. (1993) J. Biol. Chem. 268, 10029-10038) was conjugated to biotin using a di-beta-alanine spacer arm. The conjugated peptide reacted readily with the enzyme and it had an apparent Km comparable to that of the parent peptide. Using a reaction mixture consisting of 4 mg of SPA beads, 17 microM acceptor, 0.5 microM nucleotide sugar, and 7.5 U/ml enzyme, the time dependence of product formation obeyed Michaelis-Menten-type kinetics throughout the full course of the reaction-until exhaustion of the donor substrate-and the beginning portion of the reaction was sufficiently linear for calculating accurate initial rates. Analysis of the time dependency yielded an apparent Km of 0.38 +/- 0.12 microM for UDP-GalNAc. The assay is conveniently carried out in 96-well microtiter plates; it is ideally suited for assaying large numbers of samples and for screening large collections of chemicals for competitive inhibitors.

Truncation studies of alpha-melanotropin peptides identify tripeptide analogues exhibiting prolonged agonist bioactivity.

Truncation studies of alpha-melanotropin peptides identify tripeptide analogues exhibiting prolonged agonist bioactivity: PEPTIDES 17(6) 995-1002, 1996.-Systematic analysis of fragment derivatives of the superpotent alpha-MSH analogue. Ac-Ser.Tyr-Ser-Nle4-Glu- His-DPhe7-Arg-Trp-Gly-Lys-Pro-Val-NH2(NDP-MSH), led to the discovery of tripeptide agonists possessing prolonged bioactivity in the frog skin assay. Of particular significance to this discovery was Ac-DPhe-Arg-DTrp-NH2, which was the most potent tripeptide in this series exhibiting sustained melanotropic activity. Different pharmacophore models appear to exist that are dependent on the substructure and stereochemistry of the MSH(6-9) "active site." The tripeptides Ac-DPhe-Arg-Trp-NH2, Ac-DPhe-Arg-DTrp-NH2, and Ac-DPhe-DArg-Trp-NH2 stereo-chemical combinations require only Phe7-Xaa8-Trp9, whereas Ac-DPhe-DArg-DTrp-NH2, Ac-Phe-Arg-DTrp-NH2, and Ac-Phe-Arg-Trp-NH2 additionally require His4 for minimal biological activity. Ac-DPhe-Arg-DTrp-NH2 represents a novel prototype lead for the development of MSH-based peptidomimetic agonists.

Effect on protein phosphatase activity of peptide backbone modification and truncation of the autoinhibitory domain peptide of calcineurin.

Solid-phase synthesis of the autoinhibitory domain of calcineurin, CaN A467-491, also produced [aspartimide477]CaN A467-491 and [iso-Asp477]CaN467-491 when Boc-based chemistry was employed. In addition, the truncated peptide CaN A467-488 was obtained when Fmoc-based chemistry was employed. All four peptides proved to be effective inhibitors of protein phosphatase activity of calcineurin. The full-length peptide and the C-terminally truncated peptide (CaN467-488) were indistinguishable, with Ki values of 28 +/- 3 and 31 +/- 5 mu M, respectively. The internally modified peptides, [iso-Asp477]CaN A467-491 and [aspartimide477]-CaN A467-491, possessed lower inhibitory potencies (Ki values of 87 +/- 10 and 55 +/- 3 mu M, respectively).

Discovery of an alpha-melanotropin antagonist effective in vivo.

A hybrid analogue, H-His-D-Arg-Ala-Trp-D-Phe-Lys-NH2, was designed based upon the primary structures of a growth hormone-releasing peptide analogue, [His1,Lys6]GHRP, and the MSH fragment, Ac-alpha-MSH(6-11)-NH2. In vitro studies demonstrated the alpha-MSH antagonistic efficacy of the analogue in the lizards Sceloporus jarrovii and Urosaurus ornatus. In live white background-adapted S. jarrovii previously injected with the antagonist (10 nmol/5 g b.wt.), maximal skin darkening induced by alpha-MSH was reduced to 50%. In white background-adapted U. ornatus, previous injection of the analogue (1 nmol/5 g b.wt.) totally abolished the response to alpha-MSH and depressed to 50% the maximal response elicited by the superpotent MSH analogue, [Nle4,D-Phe7]alpha-MSH.

HIV protease (HIV PR) inhibitor structure-activity-selectivity, and active site molecular modeling of high affinity Leu [CH(OH)CH2]Val modified viral and nonviral substrate analogs.

This report details the structure-activity relationships of the HIV gag substrate analog Val-Ser-Gln-Asn-Leu psi[CH(OH)CH2]Val-Ile-Val (U-85548E), an inhibitor exhibiting subnanomolar affinity towards HIV type-1 aspartic proteinase (HIV-1 PR). Our data show that the P1-P2' tripeptidyl sequence provides the minimal chemical determinant for HIV-1 PR binding. We describe the structure-activity properties of Leu psi[CH(OH)CH2]Val substitution in other peptidyl ligands of nonviral substrate origin (e.g., angiotensinogen, insulin and pepstatin). Furthermore, the aspartic proteinase selectivities of a few key compounds are summarized relative to evaluation against human renin, human pepsin, and the fungal enzyme, rhizopuspepsin. These studies have led to the rational design of nanomolar potent inhibitors of both HIV-1 and HIV-2 PR. Finally, a 2.5 A resolution X-ray crystallographic structure of U-85548E complexed to synthetic HIV-1 PR dimer (Jaskolski et al., Biochemistry 30, 1600 [1991]) provided a 3-D picture of the inhibitor bound to the enzyme active site, and we performed computer-assisted molecular modeling studies to explore the possible binding modes of the above series of Leu psi[CH(OH)CH2]Val substituted HIV-1 PR inhibitors.