An alternative to conventional insect marking procedures: detection of a protein mark on pink bollworm by ELISA*

Four experiments were conducted to examine the feasibility of marking pink bollworm, Pectinophora gossypiella (Saunders) with rabbit immunoglobulin G (IgG) protein for mark-release-recapture studies. Pink bollworm were internally marked by feeding larvae an enriched rabbit IgG diet or externally marked by submerging pupae and spraying adults. Individuals were then assayed for the presence of rabbit IgG by sandwich enzyme-linked immunosorbent assay (ELISA) using anti-rabbit IgG. The internal marker was retained in larvae and retained in prepupae and pupae, but not in adults. A second experiment showed that rabbit IgG was retained on adults that were externally marked as pupae. A third series of tests examined the feasibility of externally marking adults with rabbit IgG. Rabbit IgG was retained on externally marked adults for six days in the field. Protein was retained on marked moths in the laboratory after they were captured on and removed from sticky traps. Finally, laboratory tests showed that large groups of externally marked moths transferred rabbit IgG to unmarked moths, but individual males do not readily transfer the protein to unmarked females in small vials.     

Long-distance pine pollen still germinates after meso-scale dispersal

Viability of long-distance pollen links ecological models to the genetic structure of forest tree populations, determining how forests will adapt to climate change and how far genes flow from genetically modified (GM) pine plantations. Addressing this landscape-scale inquiry is feasible when the pollen source, the delivery system, and the receiver field can be made explicit. To this end, I measured long-distance pollen germination along a 160-km transect along the North Carolina coastline, including 45000 ha of mature Pinus taeda plantations and barrier islands. Using this system, I tested three hypotheses: (1) pine pollen germinates after dispersal on meso-scale distances, (2) sodium chloride exposure reduces germination of pollen captured over open saltwater, and (3) viable pine pollen is present at high altitudes before local peak pollen shed. The experimental findings are as follows: pine pollen had germination rates of 2 to 57% after dispersal at distances from 3 to 41 km, sodium chloride solutions mildly reduced P. taeda pollen germination, and viable pine pollen grains were captured at an altitude of 610 m. GM pine plantings thus have a potential to disperse viable pollen at least 41 km from the source. Wind and rainfall, as integral parts of regional atmospheric systems, together exert a powerful influence on the genetic structure of forest tree populations.     

In vitro germination and pollen tube growth of maize (Zea mays L.) pollen

Summary Pollen grains from two hybrids, WF9xH55 (W) and K64xK55 (K) were collected and a sample from each was cultured immediately (0 h). The remainder was subdivided and stored at 2, 20, and 35° C. At 3, 6, 12, 24, 36, 48, 72, 96, and 120 h, a sample was cultured. The culture medium contained 15% sucrose, 0.6% bacto-agar, 0.03% calcium nitrate and 0.01% boric acid. Storage at 2° C resulted in a large increase in germination percentage in both W and K reaching a maximum at 24 h and then slowly decreasing with additional storage. No germination was observed at 96 h with W and at 120 h with K. The complete loss of germination occurred during a 24 h period and was very abrupt. At 20° C, a similar but less pronounced pattern was observed. However, after 24 h, aggregates of 50–1 000 pollen grains developed during storage in both W and K. Storage of W at 35° C slightly decreased the germination percentage at 3 h and eliminated it at 6 h. Storage of K at 35° C substantially increased the germination percentage at 3 h with further increases in storage periods resulting in the aggregation of grains. This general pattern of an increase at shorter storage periods followed by a gradual decrease as the storage period was extended, was found for pollen tube length and growth rate. In vitro germination characteristics can be substantially altered by the temperature and length of storage and the response to storage is associated with pollen source.Journal Series Paper No. 4566, Florida Agricultural Experiment Station.     

Reversible inhibition of the pollen germination and the stigma penetration in Crocus vernus ssp. vernus (Iridaceae) following fumigations with NO2, CO, and O3 Gases

We assessed the pollen hydration, the pollen germination, and the stigma papilla penetration of CROCUS VERNUS subsp. VERNUS (Iridaceae) after 2 h fumigations with O (3), NO (2), and CO gases within humidified (90 - 100 % RH) box experiments. When the pollen and the pistil were separately fumigated, the pollen retained the capacity to emit a tube which penetrated papilla, and the stigma papillae retained the receptivity; when the pistils were first pollinated and then fumigated, the capacity of pollen to hydrate was not affected, but the germination was significantly reduced. The vulnerability to gases became evident at 0.3 ppm O (3), 0.2 ppm NO (2), and 0.5 ppm CO. The inhibition curves as a function of the gas concentrations were of an exponential type, and they saturated at 2 ppm NO (2), 25 ppm CO, and 0.5 ppm O (3), with germination percentages of 17 %, 27 %, and 60 %, respectively. Both the pollen germination and the papilla penetration were fully restored by prolonging for 60 - 90 min the incubation at 90 - 100 % RH, after the cessation of fumigations. The vulnerability of the pollen-papilla system is discussed.     

Incompatibility and pollen competition in Alnus glutinosa: Evidence from pollination experiments

Different types of incompatibility systems were found to operate simultaneously in alnus glutinosa in the course of numerous pollination experiments, including self-pollination and pollination with controlled pollen mixtures. Isozyme genetic markers were used to identify the pollen parent of each offspring from the mixed pollination experiments, thus allowing specification of the fertilization success of each pollen parent. In a first step, these results were compared with observations on in vitro pollen germination experiments. This comparison allows for exploration of the explanatory value of different germination media as models of germination conditions on stigmas. In most cases, the data suggest that the in vitro germination conditions resemble the fertilization conditions in vivo, at least in the sense that they favor the same pollen parents. By providing a generic and operable definition of the two basic types of incompatibility, eliminating (inability to fertilize ovules) and cryptic (resulting in lowered fertilization success of a pollen parent under competition), evidence was detected for the existence of both types of incompatibility in alnus glutinosa, where eliminating incompatibility occurred as self-incompatibility only. However, since this incompatibility seems to act primarily via pollen elimination, seed production is not likely to be negatively affected in natural populations, even for comparatively large amounts of self-pollination. This revised version was published online in July 2006 with corrections to the Cover Date.     

An immuno-marking technique for thrips

Rabbit immunoglobulin G (R‐IgG) was used successfully as an external mark for thrips. Females of both Thrips tabaci Lindeman and Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) were marked with 1 mg ml^?1 R‐IgG solution with 1%‘Tween 20’ by the contact exposure method. Determining the retention of the mark was by running the rinsing solution of individual thrips in an enzyme‐linked immunosorbent assay (ELISA). The sandwich ELISA method was used with an additional biotin–avidin step. The threshold for a positive marking score was defined as three times the mean optical density readings of the negative control thrips. Under laboratory conditions, on bean pods, all marked thrips scored positive up to 6 days after marking (DAM). When marked thrips were kept in the laboratory on marigold flowers for 2 days, they all scored positive. When marked and unmarked thrips were placed together on these flowers, the mark was transferred to 10–20% of the unmarked thrips and they became positive. Under field conditions, on sticky traps covered with water‐base glue, 100, 80, and 20% of the marked T. tabaci scored positive by the 3rd, 6th, and 9th DAM, respectively. Under the same conditions 100, 90, and 10% of the marked F. occidentalis scored positive by the 3rd, 6th, and 9th DAM, respectively. The retention of the R‐IgG decreased significantly under conditions of wetness and high humidity. After 6 days on chive plants kept at 80–100% r.h., all marked thrips scored negative while on plants kept at 40–60% r.h., 85% of the marked thrips scored positive. Rabbit IgG can be used as an external marker for thrips. The suitability of this marking method for dispersal studies of these important pests needs to be evaluated.     

A Common Marking Technique Affects Tadpole Behavior and Risk of Predation

In many studies, it is necessary for researchers to mark individual animals for later identification. It is often assumed in the interpretation of these studies that marks have no effects on the biology of the animals. This assumption is insufficiently tested, and, when it is, coarse assessments of negative effects are often used, such as survival and growth under simplified laboratory conditions. We examined the consequences of a common larval amphibian marking technique (staining with methylene blue) for wood frog tadpole behavior and survival in an ecologically realistic scenario (predation). We measured a number of tadpole behavioral variables, under baseline conditions and in the presence of olfactory cues of a predator, for marked and unmarked tadpoles. We then exposed pairs of tadpoles (one marked and one unmarked) to one of two predators and tested for the effects of marking on the susceptibility of tadpoles to predation. We found that marking suppressed the increase in movement rate that typically occurred in (unmarked) tadpoles in the presence of predator cues. Marked tadpoles were significantly more likely to be captured by predators, an effect that could not be attributed to this difference in movement rate. These results raise concern about the use of this staining method and highlight the need for studies involving marked animals to thoroughly address any relevant effects the marks may have on the biology of the subjects.     

Pollen-limited seed set in pseudogamous blackberries (Rubus L. subgen. Rubus)

Summary Pseudogamous blackberry species are polyploid and usually exhibit meiotic irregularities causing severe reduction in pollen viability. When species means were compared, relative seed set was strongly correlated with the number of good pollen grains produced per flower divided by the number of ovules (good-pollen/ovule ratio).Partial correlation analysis for percentage viable pollen and relative seed set, controlling for good-pollen/ovule ratio, revealed an almost significant relationship, presumably because meiotic irregularities have a similar effect on both pollen viability and the viability of meiotically derived embryo sacs. Seed germination, on the other hand, showed no relationship with relative seed set or with pollen viability.The automatic selfing capacity exhibited by most of these blackberry species does not appear to be related to either pollen production or relative seed set.     

RELATIVE SUCCESS OF SELF AND OUTCROSS POLLEN COMPARING MIXED- AND SINGLE-DONOR POLLINATIONS IN AQUILEGIA CAERULEA

Flowers frequently receive both self (S) and outcross (OC) pollen, but S pollen often sires proportionally fewer seeds. Failure of S pollen can reflect evolved mechanisms that promote outcrossing and/or inbreeding depression expressed during seed development. The relative importance of these two processes was investigated in Aquilegia caerulea, a self-compatible perennial herb. In the field I performed single-donor (S or OC) and mixed-donor (S plus OC) pollinations to compare the relative success of both pollen types at various stages from pollen germination to seed maturity. Single-donor S pollinations produced significantly fewer and lighter seeds (x decrease = 12% and 3%, respectively) than OC pollinations. Abortion rates differed by an average of 38% whereas fertilization rates differed by only 5%, indicating that most differences in seed number arose postzygotically. This suggests that inbreeding depression was responsible for most failure of S pollen. One prezygotic effect measured was that 10% fewer S than OC pollen tubes reached ovaries after 42 hr, suggesting S pollen might fertilize proportionately fewer ovules after mixed pollination. Using allozyme markers, I found mixed-donor pollinations produced significantly more and heavier outcrossed than selfed seeds. However, the proportion of selfed seed, fertilized ovules, and aborted seeds for mixed-donor fruits were each predictable from pollen performance in single-donor fruits, suggesting that differential paternity is best explained by inbreeding depression during seed development. Even given these similarities between mixed- and single-donor fruits in the relative performance of S and OC pollen, both individual seed weight and seed set were significantly higher in multiply-sired fruits.     

Individuals' behaviors following dye-marking in wild black-and-white colobus (Colobus vellerosus)

The ability to recognize individuals is a prerequisite for analyzing social relationships. We marked five adult and subadult Colobus vellerosus (three in 2002, and two in 2003) at the Boabeng Fiema Monkey Sanctuary, Ghana, to assess the feasibility of dye-marking black-and-white colobus, describe their reactions, and compare some of their behaviors with those of unmarked individuals. We used Nyanzol-D, a nontoxic black dye sprayed on the white tail (or white thigh) of the animal with a spray gun or a tree sprayer. Reactions to the marking procedure ranged from moving away and staring at the observer, without interruption in feeding (in one subject), to fleeing about 5 m away (in four subjects). In 234 hr of ad libitum observations (in 2002 and 2003), marks were scratched or otherwise were the object of attention from the bearer or other individuals on only one occasion. In 2002 we collected 22 hr of observations on the three marked monkeys and some unmarked monkeys in 10-min focal samples. Neither the marked nor the unmarked animals attended to the marks during focal samples. Marked and unmarked individuals displayed similar rates of displacement activities (autogrooming, scratching, and yawning). The proportion of scans with at least one near neighbor varied between marked and unmarked subjects, but the direction of the difference was not the same between males and females. The only aggression observed was displacements, and only in one comparison (out of four) did a difference emerge: the marked subadult male received more displacements than the unmarked males. Overall, marked and unmarked individuals did not differ consistently in our measures. Examination of the potential effects of marking should continue, since changes in pelage coloration may have longer-term social effects in species that rely largely on vision.     

Ovule pre-emption and pollen limitation in a self-fertile perennial herb (Blandfordia grandiflora,Liliaceae)

The extent, frequency and causes of pollenlimited seed production were examined in partially selffertile populations of Blandfordia grandiflora for 2 years. Percentage seed set of open-pollinated plants (50–57%) did not differ within or between years, and was about 19% less than experimentally cross-pollinated plants (70–75%). Floral visits by honeybees did not differ through the flowering season and the number of pollen grains deposited on stigmas within 12 h of flowers opening exceeded the number of ovules per flower, indicating that the quality rather than the quantity of pollination limited seed set. Pollen limitation was caused by concurrent self- and cross-pollination and the subsequent abortion of some selfed ovules due to inbreeding depression. Natural seed set (55%) was intermediate between selfed (43%) and crossed (75%) flowers and was not increased when flowers that had been available to pollinators for 24 h were hand cross-pollinated, suggesting that ovules were already fertilized. Similarly, experimental pollination with both cross and self pollen within 24 h of flowers opening did not increase seed set relative to natural seed set, indicating that both cross- and self-fertilizations had occurred. In contrast, when selfing followed crossing by 48 h, or vice versa, seed set did not differ from crossed-only or selfed-only flowers, respectively, indicating that ovules were pre-empted by the first pollination. Collectively, these results indicate that under natural conditions self pollen pre-empts ovules, rendering them unavailable for cross-fertilization. This selfing reduces fecundity by 50%, as estimated from the natural production of cross seeds when selfing was prevented. Consequently, selection should favour floral traits, such as increased stigma-anther separation or protandry, that reduce interference between male and female functions that leads to selfing.     

Aerated hydration treatment for improved germination and seedling growth in aubergine (Solarium melongena) and pepper (Capsicum annuum)

Aerated hydration treatment of aubergine and pepper seeds at 25°C (42 h for aubergine, 48 h for pepper) followed by drying increased percent germination across a range of test temperatures (18°C, 25°C and 35°C). The increase in germination obtained by the treatment was retained in storage for up to 4 months. In a glasshouse experiment, aerated hydration decreased mean emergence time but increased seedling dry weight in both species and decreased the proportion of abnormal seedlings in pepper. This work suggests that aerated hydration treatment can improve establishment of these crops in a range of planting temperatures.     

In vitro pollination of isolated ovules of Cichorium intybus L.

 A method for the in vitro pollination of chicory (Cichorium intybus L.) ovules was developed. The purpose was to avoid self-incompatibility after in vitro self-pollination of ovules isolated from flower buds before anthesis and from open flower heads. Seedlings were obtained at a low percentage (0.76%), and the results were explained in terms of pollen viability, pollen germination on the ovule, embryogenesis studies and ploidy analysis. Received: 9 April 1997 / Revision received: 23 July 1997 / Accepted: 6 September 1999     

Effects of radiotransmitters and plasticine bands on Mallard duckling survival

ABSTRACT Individual markers are essential for measuring survival rates, but few reliable techniques exist for marking young waterfowl. We banded 572‐d‐old Mallard (Anas platyrhynchos) ducklings from 85 broods with plasticine‐filled leg bands in 2006–2007 and marked 82 hatchling Mallards from 43 broods with 1.8‐g back‐mounted prong‐and‐suture transmitters in 2006. We compared survival probabilities of marked ducklings and 621 unmarked ducklings from 110 broods where only the female was radio‐marked using the nest survival module in Program MARK. The best‐supported model recognized deleterious effects of both radiotransmitters (β=?0.82, 85% CI =?1.32 to ?0.33) and plasticine bands (β=?0.29, 85% CI =?0.55 to ?0.03). Cumulative survival to 30 d of age for unmarked ducklings averaged 0.22 (process variation; σ= 0.11) in 2006 and 0.20 (σ= 0.11) in 2007, whereas 30‐d survival of banded ducklings during these 2 yr was 0.14 (σ= 0.09) and 0.13 (σ= 0.09), respectively. Radio‐marked ducklings averaged 0.04 (σ= 0.03) cumulative survival in 2006. Although capturing ducklings after they leave nests is not always possible, post hoc analysis showed that marker effects decreased with duckling age and waiting until ducklings were at least 4 d old before marking would have negated these effects. Because marking hatchling Mallards with either radiotransmitters or plasticine bands negatively impacted their survival, we do not recommend these marking techniques for newly hatched ducklings.     

Microdot technology for individual marking of small arthropods

• 1 Individual mark–release–recapture is an important method for gathering data on insect movement, although it is limited by the constraints of tagging small insects with individual information.
• 2 Microdots, originally developed for covert security applications, are small polymer discs (diameter 0.5 mm) bearing up to 26 characters of information and have the potential as an alternative to the larger bee tags. In the present study, we test microdots for the individual marking of a 9‐mm parasitoid wasp.
• 3 We individually marked 505 wasps. The recapture rate was 24% of individuals over 189 recapture events, for which 84% retained legible microdot labels. Movement was in the range 0–161 m with a mean displacement 21.2 ± 2.7 m. A captive survival experiment showed no difference in lifespan between marked and unmarked wasps.
• 4 The present study shows that microdots can provide an effective, durable, low‐cost method for individually tagging small insects. The technique offers new opportunities by greatly expanding the capability for individually marking small insects, shifting the minimum size below that of bee tags, which is the only other manufactured option for individualized miniature marking.

     

Effects of high humidity and temperature stress on pollen membrane integrity and pollen vigour in Nicotiana tabacum

Summary The prolonged exposure of pollen Nicotiana tabacum to high humidity at both room temperature and 38° C did not affect membrane integrity as revealed by the fluorochromatic reaction (FCR) test, but did affect pollen vigour. At room temperature germination was not affected, although tube growth was reduced; at 38° C, there was both a reduction in tube growth and delayed germination. When the pollen was subjected to 1 h hydration followed by 1 h desiccation (up to a maximum of four cycles) at room temperature, a reduction in the FCR, germination and tube length after each desiccation treatment was observed. Subsequent hydration fully restored the FCR, but only partially restored germination and tube growth. At 38° C, however, FCR, germination, and tube growth were drastically reduced. The implications of these results on the relationship between FCR and germinability, the responses of pollen exposed to humidity and temperature stress in the field, and on pollen storage are discussed.     

REPRODUCTIVE COSTS OF SELF-POLLINATION IN IPOMOPSIS AGGREGATA (POLEMONIACEAE): ARE OVULES USURPED?

In common with many cosexual angiosperms, the hummingbird-pollinated montane herb Ipomopsis aggregata (Polemoniaceae) is likely to experience self-pollination. Does this incur a fecundity cost even in such a highly self-sterile (presumably self-incompatible) species? Histological studies showed that self pollen germinates, and its tubes penetrate ovules almost as often as those of outcross pollen. Ovules penetrated by self tubes are especially likely to show milky callose occlusion 24–48 hr after pollination, and several observations suggest an association between occlusion and degeneration. Compared to flowers receiving only outcross pollen, seed set was reduced by 42% on average when self pollen was applied along with outcross, either by hand (in emasculated flowers) or by natural autodeposition (in unemasculated flowers). Reductions were statistically indistinguishable whether self pollen was applied 9 hr before outcross pollen, or at the same time. Unemasculated flowers accumulated substantial self pollen loads, and this autodeposition persisted when flowers were probed to mimic hummingbird visitation. Geitonogamy also is substantial, judging from field estimates of pollen transfer. Thus natural self pollen deposition may be sufficient to “usurp” ovules that otherwise could mature. In this light, late-acting self-rejection in I. aggregata seems decidedly less efficient than an early-acting system that would block pollen germination or tube growth.     

Marking pike fry otoliths with alizarin complexone and strontium: an evaluation of methods

Laboratory experiments demonstrated that both alizarin complexone and strontium are useful in mass marking of pike Esox lucius fry otoliths. Visual detection of alizarin complexone marks was considered more reliable than the quantitative analysis of strontium for differentiating marked and unmarked individuals after release in a Danish lake.