Effect of cryopreservation on the pre-hatching behavior in the Mexican fruit fly Anastrepha ludens Loew (Diptera, Tephritidae).

In a sampling of untreated embryos of the economically important fruit pest species, Anastrepha ludens, the cumulative hatch percentage in the lab was noted to be ∼85%. Approximately 70% of the larvae had eclosed through the posterior pole of the egg. This process is effected by the act of Pole Reversal (PR) of the fully developed pre-hatch larva from the wider anterior to the narrower posterior pole of the egg. Investigation of the effects of cryopreservation and various pretreatments prior to cryostorage on the PR behavior was prompted by the observation of significantly lower proportion of cryopreserved embryos exhibiting the PR behavior. Pretreatments (dechorionation and permeabilization) followed by vitrification resulted in delayed hatching, reflecting a slower embryonic development rate of ∼10 h. A smaller proportion of the treated embryos either eclosed from the anterior end of the egg or did not eclose at all despite complete development and prehatch gnawing activity. In the untreated controls, 24.0% of the embryos eclosed from the anterior pole. After permeabilization and cryopreservation, 83% and 55% (adjusted hatch) of the embryos were noted to hatch this way, respectively. An analysis of the hatch count after the treatments shows that factors contributing to the embryos' inability to properly invert polarity is not solely due to cryopreservation but also due to the pretreatment procedures including dechorionation and permeabilization. In fact, the permeabilization pre-treatment contributed the highest to this phenomenon lending support to the view that chemical toxicity rather than physical effects of cryopreservation play a major role in post-cryopreservation effects.

Stage selection and restricted oviposition period improves cryopreservation of dipteran embryos.

Embryos of two dipteran species (Musca domestica and Lucilia sericata) were assessed for an effective sampling time that would result in the highest post-cryopreservation hatch rate, with a primary goal to define species-specific egg collection periods and the effects of manual stage selection on post cryopreservation yield. The effects of the time taken to collect eggs on, (a) the proportion of embryos reaching a specific developmental stage between 17 and 20 h of development, and (b) the post-cryopreservation hatch rate were assessed. Permeabilization treatment applied at any stage of embryonic development did not significantly reduce embryo viability. Eggs collected over longer durations significantly reduced the number of embryos available in a specific developmental stage amenable to cryopreservation. Hatch percentage after cryopreservation of the embryos of M. domestica collected over a 60 min period was 10.7 ± 8.7% compared to 31 ± 5% for the eggs collected for just 15 min. Similarly, percent hatch in L. sericata resulted in 17.0 ± 3.9 and <2% for 15 and 60 min samples, respectively. Significantly higher hatching rates were obtained for cryopreservation after manual selection of specific embryonic developmental stages from the dechorionated samples. Post-cryopreservation hatching rate for stage-selected M. domestica embryos was 86.5 ± 5.5% compared to 33.3 ± 4.5% for embryos staged only by an overall visual confirmation. In the case of L. sericata, the hatching percentage was 79.0 ± 11.1 for stage-selected embryos compared to 17.0 ± 3.9% without individual selection.

Cryopreservation of embryos of Lucilia sericata (Diptera: Calliphoridae).

Embryos of Lucilia (Phaenicia) sericata (Meigen) (Diptera Calliphoridae), the green blowfly, were successfully cryopreserved by vitrification in liquid nitrogen and stored for 8 yr. Embryos incubated at 19 degrees C for 17 h after oviposition were found to be the most appropriate stage to cryopreserve. Removal of the embryonic surface water was done using 2-propanol before the alkane treatment to permeabilize the embryo. Exposure to 2-propanol for > 10 s caused necrotic tissue damage in the embryos. Among the alkanes used, hexane was found to be a superior permeabilizing solvent compared with heptane or octane, with embryo hatching rates on par with the controls. Treatment with the vitrification solution for < 12 min was insufficient to vitrify the embryos. Treatment time in the solution beyond 15 min reduced embryo viability. However, the percentage of embryos vitrifying upon exposure to liquid nitrogen vapor remained constant after 12 min of treatment. Long-term storage was initiated in 2004, and the mean hatch percentage recorded then for the short-term cryopreserved embryos was 9.51%. When the long-term stored samples were retrieved in 2012, 8.47% of the embryos hatched, 66.36% larvae pupariated, and 36.96% of the pupae eclosed. Recent optimization of the technique has resulted in a hatch rate of 34.08 +/- 15.5%, of which 67.5% of the larvae pupariated and 72% of the pupae eclosed to normal flies.

Permeability barriers to embryo cryopreservation of Pectinophora gossypiella (Lepidoptera: Gelechiidae).

The aim of this study was to develop a method to cryopreserve the embryos of the pink bollworm moth, Pectinophora gossypiella (Saunders). Previously developed dipteran cryopreservation protocols were not directly adaptable to use with the embryos of this lepidopteran species. Physiochemical and electron microscope observations revealed substantial differences in the structure of the chorion, wax layer, and vitelline membrane complex when comparing the cryopreservable embryonic stages of P. gossypiella and dipteran embryos. Thus, the initial steps dealing with dechorionation and permeabilization were ineffective and had to be altered. Exposure to the sodium hypochlorite-based chorion removal step decreased P. gossypiella embryo viability to a very low level. Survival increased and permeability was evident when an alkane wash was used as the first step in the procedure. After the alkane treatment with a surfactant yielded the maximum exchange of cryoprotectant with water as evidenced by a significant lowering of the supercooling point of the cryoprotectant-loaded embryos. The remainder of the cryopreservation and storage recovery protocol for P. gossypiella was similar to those developed for dipteran embryos. Survival of recovered, hatched embryos to adulthood was approximately 7%.

Cold storage of Culex pipiens in the absence of diapause.

A major expenditure in vector biology laboratories is the rearing of mosquitoes. Most mosquito colonies require substantial effort to maintain, including frequent bloodmeals for optimal performance. Successful cryopreservation of mosquitoes continues to be elusive. Although using diapause as a storage mechanism is an option for mosquito preservation, several obstacles include the lack of a well-characterized diapause or the inability of some species to enter diapause. Thus, other options for preservation are needed. To address this issue, we investigated the use of long-term low-temperature storage in the absence of diapause for adults of the northern house mosquito, Culex pipiens L. Our results indicate that although male longevity is not substantially increased by cold storage, female longevity is dramatically increased by storage at lower temperatures. When mated before storage, females remain reproductively viable after at least 10 wk of storage, although at reduced levels. These results indicate that cold storage without diapause induction is a viable option for colony maintenance in vector biology laboratories.

Cold storage of adult Gonatocerus ashmeadi (Hymenoptera: Mymaridae) and effects on maternal and progeny fitness.

Storage of Conatocerus ashmeadi Girault (Hymenoptera: Mymaridae) adults at 2, 5, and 10 degrees C showed that these parasitoids do not survive at 2 degrees C for 5 d, and exposure to 5 and 10 degrees C shortens their life span. The lethal time (LT) 50 (i.e., length of storage time for 50% wasp survival) at 5 degrees C was 14 d for males and approximately 29 d for females, whereas at 10 degrees C was 32 and 39 d, respectively. Effects of adult storage at 10 degrees C on other factors indicating fitness, such as fecundity, developmental time, parasitism, emergence, and sex ratio, were examined on female wasps and their progeny at 10-d intervals for up to 60 d. Glassy-winged sharpshooter, Homalodisca vitripennis (Germar), eggs were used as hosts for propagation of this wasp and for assessing its oviposition and fecundity. Increasing adult storage time decreased the length of the ovipositional period for the maternal generation, and oviposition was decreased by 90% after 60-d storage. A significant reduction in maternal lifetime fecundity occurred after 20-d storage and in the incidence of parasitism after 40 d. We also found a carryover effect caused by storage of the maternal generation that was expressed in the F1 generation. When cold storage of the adult parents was > or = 20 d, we observed delayed development, decreased fecundity, reduced longevity, and increased male production occurring in the F1 generation. Reduced fitness of the F1 generation was also expressed as a decrease in net reproductive rate (R(o)) and an increase in mean generation time (T(c)). However, none of these deleterious effects were evident in the F2 progeny that descended from grandparents that had experienced cold storage. Damage caused by indirect chilling injury and/or induced maternal aging occurring during storage can account for the decreased fitness of maternal and F1 generations. Providing that the limits of cold tolerance of G. ashmeadi as defined in this study are not exceeded, our results show that short-term cold storage of adults could be used in a mass-rearing program.

Progeny quality of Gonatocerus ashmeadi (Hymenoptera: Mymaridae) reared on stored eggs of Homalodisca coagulata (Hemiptera: Cicadellidae).

This study assessed the effects of refrigerated storage on the suitability of eggs of the glassy-winged sharpshooter, Homalodisca coagulata (Say) (Hemiptera: Cicadellidae), as hosts for propagation of the parasitoid Gonatocerus ashmeadi Girault (Hymenoptera: Mymaridae). Development of the host eggs was terminated by chilling at 2 degrees C for 5 d before storage was initiated at 10 degresC for up to 70 d. Parasitism, adult emergence rate, developmental time, and sex ratio were used to gauge the suitability of the eggs as hosts after storage. In addition to these measures, demographic growth parameters also were used to assess the quality of the wasp progeny through the F2 generation. Host eggs stored 20 d remained fully acceptable to the wasps for attack. Although the parasitism rate decreased with storage time, > 80% adult parasitoid emergence was realized from eggs stored 30 d. After 70 d storage, adult emergence rate was decreased by 48%, fecundity decreased by 53%, female production by 19%, developmental time was extended 3 d, and female longevity was shortened 5 d. The emergence pattern of F1 but not F2 adults varied with storage time of the parental and grandparental hosts, respectively. For the F1 generation, emergence rate, development, and sex ratio did not vary with storage time when the F1 parents parasitized fresh host eggs. Demographic parameters for the F, population showed that net reproductive rate was > 20 although it decreased significantly after their parental host eggs were stored for > 30 d. The intrinsic and finite rates of increase, population doubling time, and mean generation time decreased only after storage for 60 d. Our results show that short-term cold storage could be used for maintaining wasp populations in a mass-rearing program and that the detrimental effects of chilling host eggs in storage for over 30 d do not extend to F2 generation.

Composition of the surface hydrocarbons from the vitelline membranes of dipteran embryos.

Hydrocarbons were the major lipid class extracted by hexane from the vitelline membrane surface of dechorionated eggs of the house fly, Musca domestica, the New World screwworm, Cochliomyia hominivorax, the secondary screwworm, Cochliomyia macellaria, the green bottle fly, Phaenicia sericata, the sheep blow fly, Lucilia cuprina and the Mexican fruit fly, Anastrepha ludens. The length of time the embryos must be exposed to hexane with or without a small amount of alcohol in order to attain permeability was species-dependant. Long-chain n-alkanes comprised the major lipid class removed from vitelline membranes of all species except A. ludens where 2-methylalkanes were the major class. The range in size by the total number of carbon atoms in the hydrocarbons was: C23-C49 in C. hominivorax, C27-C33 in C. macellaria, C24-C35 in L. cuprina, C25-C36 in M. domestica, C25-C33 in P. sericata and C21-C51 in A. ludens. The major hydrocarbon component, expressed as percent of the total hydrocarbons, was n-nonacosane (C29) in C. hominivorax (40%), C. macellaria (43%), L. cuprina (38%), M. domestica (39%) and P. sericata (60%). However, in A. ludens, 2-methyloctacosane (32%) was the major hydrocarbon. Unsaturated hydrocarbons, monoenes (16%) and dienes (11%), were abundant only in A. ludens. Since prior studies indicated that the length of time the embryos must be exposed to hexane with or without a small amount of alcohol in order to attain permeability is species dependant, we suggest that the differences in hydrocarbon composition may contribute to this variation in lipid extractability.

Host plant effects on development and reproduction of the glassy-winged sharpshooter, Homalodisca vitripennis (Homoptera: Cicadellidae).

Development, survivorship, longevity, reproduction, and life table parameters of the glassy-winged sharpshooter, Homalodisca vitripennis (Germar), were examined in the laboratory using three host plants, sunflower (Helianthus annuus L.), Chrysanthemum morifolium L., and euonymus (Euonymus japonica Thurb.). Females deposited similar-sized egg masses on all three plants. Hatching was highest with eggs deposited on euonymus and lowest for those deposited on sunflower. Embryonic development time among host plants was similar while nymph development time was shortest on sunflower and longest on euonymus. Nymph survival to adulthood ranged from 32% on euonymus to 82% for those reared on sunflower. Adult females had similar life spans on sunflower and chrysanthemum. H. vitripennis completed a lengthy egg-to-adult development on euonymus, however, mating did not occur. The onset of mating was contingent on maturation of adult females. The majority of mating activity occurred within the first three days after onset. Premating periods ranged from 6 to 7 d on sunflower to 27 d on chrysanthemum, with overall mating rates of 77.4 and 19.8%, respectively. Females typically mated more than once and they had the longest oviposition period and highest egg production on sunflower; ≈ 50 and 67% of total number of eggs were deposited within first 45 d after the start of oviposition on sunflower and chrysanthemum, respectively. Adult size and weight related to which host plant was consumed throughout development. Greater intrinsic and finite rates of increase and net reproduction rate, and shorter population doubling time occurred when the sharpshooters were allowed to develop on sunflower. The overall developmental and reproductive parameters obtained in this study indicate that a mixed host plant system, composed of sunflower and euonymus or chrysanthemum plants, is an efficient means for optimizing egg production and colony maintenance of the glassy-winged sharpshooter.

Mouthpart morphology and stylet penetration of host plants by the glassy-winged sharpshooter, Homalodisca coagulata, (Homoptera: Cicadellidae).

The ultrastructural morphology of the mouthparts of the glassy-winged sharpshooter, Homalodisca coagulata, and method of plant penetration was examined using light microscopy, scanning electron microscopy, and transmission electron microscopy methods. The gross morphology of the labrum, labium, and stylet fascicle was consistent with what has been described for other plant-sucking homopterans. The ultrastructural examination of the mouthparts revealed unique details that have previously gone unreported. Several types of sensilla-like structures having the form of pegs and multi-lobed objects were identified on the outer surfaces of the labrum and within the labial groove. Dendritic canals terminated in an extensive network of smaller canals at the distal tip of the maxillary stylets below a series of surface denticles suggesting that this area may have a sensory function associated with locating xylem elements of host plants. Examination of salivary sheath pathways established that 65% of the plant penetrations by this insect terminated in the xylem vessels of the host plant. Probing by the insect was largely intracellular and terminal branching of a single probe site was common. Plant surface feeding sites varied with the stage of development which correlates with the depth of the xylem vessels and the length of the maxillary stylets of the various instars.