Hooked setae: tests of the anchor hypothesis

Abstract. We tested the hypothesis that hooked setae function as anchors in three species of tubiculous polychaetes ( Eudistylia vancouveri, Schizobranchia insignis , and Owenia fusiformis ). All maintained position within their tubes when exposed to high pressures (up to 100–200 kPa) applied from the posterior direction (where it would tend to cause the tips of hooks to embed in the tube wall). When pressure was applied in the opposite direction, where hooks would not tend to embed in the tube wall, the worms were expelled from their tubes at lower pressures (30–100 kPa). The ability of these worms to maintain their position within their tubes was independent of body size. On the basis of these findings we made three predictions. First, worms that use their hooked setae as anchors should have those hooks located on the body in greatest number and size on the segments associated with greatest worm diameter. Second, as worms increase in size, setal armory should increase in a predictable way. The force that can be applied to extract worms from their tubes by suction feeding fish or wave action would increase as the area subject to suction increases (proportional to the cross sectional area of the tube). Therefore, we predict that setal armory should also increase as a squared function. Third, hooks or uncini should show patterns of wear or loss and/or the worms' bodies should show scars or wounds where the setae are most used (e.g., where worm diameter is at its maximum). All of these predictions were supported by the data and indicate that hooked setae function as anchors for tubiculous polychaetes. This is important for understanding the biology of these animals and has implications for using hooked setae as characters in phylogenetic analyses.     

Commensal symbiosis between agglutinated polychaetes and sulfate‐reducing bacteria

Pendant bioconstructions occur within submerged caves in the Plemmirio Marine Protected Area in SE Sicily, Italy. These rigid structures, here termed biostalactites, were synsedimentarily lithified by clotted‐peloidal microbial carbonate that has a high bacterial lipid biomarker content with abundant compounds derived from sulfate‐reducing bacteria. The main framework builders are polychaete serpulid worms, mainly Protula with subordinate Semivermilia and Josephella. These polychaetes have lamellar and/or fibrillar wall structure. In contrast, small agglutinated terebellid tubes, which are a minor component of the biostalactites, are discontinuous and irregular with a peloidal micritic microfabric. The peloids, formed by bacterial sulfate reduction, appear to have been utilized by terebellids to construct tubes in an environment where other particulate sediment is scarce. We suggest that the bacteria obtained food from the worms in the form of fecal material and/or from the decaying tissue of surrounding organisms and that the worms obtained peloidal micrite with which to construct their tubes, either as grains and/or as tube encompassing biofilm. Peloidal worm tubes have rarely been reported in the recent but closely resemble examples in the geological record that extend back at least to the early Carboniferous. This suggests a long‐lived commensal relationship between some polychaete worms and heterotrophic, especially sulfate‐reducing, bacteria.     

Chaetae and mechanical function: tools no Metazoan class should be without

To address the functional contributions of capillary chaetae in the maldanid polychaete Clymenella torquata, we compared irrigation efficiency and tube structure for animals with intact and trimmed capillary chaetae. We measured pumping rates for worms before and after they were anaesthetized and subjected either to capillary trimming or mock trimming, i.e. handling without trimming. Worms with trimmed chaetae were significantly less effective at moving water through their tubes than those with intact chaetae. There were no significant differences in the ability of control worms to move water within their tubes. No significant changes in rates of peristalsis were observed among experimental or control groups. These data strongly suggest that body musculature and capillary chaetae work in concert to hold worms in position within tubes during peristaltic pumping. When chaetae are shortened, the body musculature must contract to a greater degree, increasing the functional diameter of the worm to achieve the necessary traction with the tube wall, resulting in less efficient irrigation. We also compared the inner diameters of original field tubes to tubes built by control worms or worms after capillary trimming. The inner diameters of new tubes built by worms with shortened chaetae were larger than their original tubes, while those of both control groups were not. One possible explanation is that the chaetae have a sensory role and shortened chaetae send the false message that the nascent tube walls are farther away than they are, the body contracts in compensation and the tube is widened, however this idea has not been tested.     

Potential influence of tubicolous worms on the bottom roughness length z0 in the south-western Baltic Sea

The influence of three different species of tubicolous worms (Pygospio elegans, Polydora ciliata and Lagis koreni) on the hydrodynamic bottom roughness length (z₀) was analysed in this study. Flume experiments and geospatial methods were combined to determine the potential interactions between worm tubes and the near-bed flow regime and the resulting effects on sediment transport in the south-western Baltic Sea. The three selected species are common in the area of interest (3539 km²). Their species-specific population densities were taken from existing macrozoobenthos datasets and transferred into a Geographic Information System (GIS). In analogy to the sediment roughness length, the hydrodynamic roughness lengths generated by the tubicolous worms were calculated and corresponding sediment transport values, derived from flume experiments with artificial tube lawns, were geospatially analysed using GIS. In order to show the direct influence of worm tubes on the surrounding sediment surface flume experiments were conducted at two given current velocities of 20 cm s^− 1 for sediment displacement effects and 5 cm s^− 1 for deposition effects. The roughness length was shown to increase by a factor of 2 to 30 in the presence of biogenic structures such as the worm tubes. The near-bed hydrodynamic conditions are significantly influenced at low roughness densities through independent or isolated flow conditions at 0.7 to 1.9% and at high roughness densities between 4.2 and 7.5%, resulting in unaffected sediment surfaces through “skimming flow”, as well. The GIS analysis revealed that this effect occurs over 4% (137 km²) of the area of investigation, whereas sediment displacement at roughness densities between 0.7 and 1.9% due to increased turbulence is the predominant effect over 33% (present on 1172 km²) of the area of investigation. These findings reveal the important influence of species-generated microtopography on sediment transport processes.     

Microridges in Cyprinus carpio scale epidermis

Actin‐based microridges were evaluated in koi scale epidermis in situ. The fingerprint‐patterned microridges covered the dorsal face of superficial layer cells and were overall similar to that described in many fishes. Several other microridge patterns were observed, however, ranging from loose or tightly packed ridges, fragmented ridges, a honeycomb ridge pattern and the presence of actin‐rich puncta. Individual F‐actin‐stained microridges varied greatly in length, from a few to 30 μm or more, with a few single ridges extending the entire perimeter of a cell. Branched microridges, comprised of single ridges that appeared continuous with each other, extended to over 150 μm in some cases. The actin‐binding proteins α‐actinin and cortactin were distributed in a dot‐like pattern along the length of individual ridges, consistent with bundled actin cores described in earlier studies. Antiphosphotyrosine antibody failed to detect this signal transduction‐related amino acid modification in microridges unless tyrosine phosphatases were first inhibited, after which bright phosphotyrosine‐rich dots were detected along the microridges.     

The pollen tube: a soft shell with a hard core

Plant cell expansion is controlled by a fine‐tuned balance between intracellular turgor pressure, cell wall loosening and cell wall biosynthesis. To understand these processes, it is important to gain in‐depth knowledge of cell wall mechanics. Pollen tubes are tip‐growing cells that provide an ideal system to study mechanical properties at the single cell level. With the available approaches it was not easy to measure important mechanical parameters of pollen tubes, such as the elasticity of the cell wall. We used a cellular force microscope (CFM) to measure the apparent stiffness of lily pollen tubes. In combination with a mechanical model based on the finite element method (FEM), this allowed us to calculate turgor pressure and cell wall elasticity, which we found to be around 0.3 MPa and 20–90 MPa, respectively. Furthermore, and in contrast to previous reports, we showed that the difference in stiffness between the pollen tube tip and the shank can be explained solely by the geometry of the pollen tube. CFM, in combination with an FEM‐based model, provides a powerful method to evaluate important mechanical parameters of single, growing cells. Our findings indicate that the cell wall of growing pollen tubes has mechanical properties similar to rubber. This suggests that a fully turgid pollen tube is a relatively stiff, yet flexible cell that can react very quickly to obstacles or attractants by adjusting the direction of growth on its way through the female transmitting tissue.     

Unique mating behavior,and reproductive biology of a simultaneous hermaphroditic marine flatworm (Platyhelminthes,Tricladida, Maricola)

Flatworms generally are simultaneous hermaphrodites that exhibit various kinds of mating behavior. Here we report on the mating behavior and reproductive biology of the planarian Paucumara falcata. We recognized three phases in its mating behavior: a courtship, copulation, and postcopulatory phase. During the last‐mentioned phase, the partners showed a unique and very characteristic behavior in which their bodies intertwined, forming a spiral. Histological study of partners in copula revealed that the sclerotic tip of the musculo‐parenchymatic organ pierces the body wall of the partner and then becomes lodged in its parenchyma, suggesting that this organ may act as an anchor, thus stabilizing the worms during copulation. Similar organs in other species of marine triclad may also perform a stabilizing role during copulation. During copulation in individuals of P. falcata, sperm transfer was reciprocal or only unilateral. Copulation duration ranged 13–35 min (average 20 ± 5 min), irrespective of whether the mating was successful (i.e., resulted in the production of fertile cocoons). The spiraling phase lasted on average 10 min; some worms did not show the postcopulatory spiraling phase during their mating behavior. After successful copulation, an individual worm produced 1–12 fertile cocoons over a period of 1–17 days; from a cocoon hatched either one young (in 70% of the cases), or two young worms.     

An Ultrastructural Study of the Extruded Polar Tube of Anncaliia algerae (Microsporidia)

All microsporidia share a unique, extracellular spore stage, containing the infective sporoplasm and the apparatus for initiating infection. The polar filament/polar tube when exiting the spore transports the sporoplasm through it into a host cell. While universal, these structures and processes have been enigmatic. This study utilized several types of microscopy, describing and extending our understanding of these structures and their functions. Cryogenically preserved polar tubes vary in diameter from 155 to over 200 nm, noticeably larger than fixed‐sectioned or negatively stained samples. The polar tube surface is pleated and covered with fine fibrillar material that projects from the surface and is organized in clusters or tufts. These fibrils may be the sites of glycoproteins providing protection and aiding infectivity. The polar tube surface is ridged with 5–6 nm spacing between ridges, enabling the polar tube to rapidly increase its diameter to facilitate the passage of the various cargo including cylinders, sacs or vesicles filled with particulate material and the intact sporoplasm containing a diplokaryon. The lumen of the tube is lined with a membrane that facilitates this passage. Careful examination of the terminus of the tube indicates that it has a closed tip where the membranes for the terminal sac are located.     

Ultrastructure of feeding tubes formed in giant-cells induced in plants by the root-knot nematodeMeloidogyne incognita

Summary The plant pathogenic nematodeMeloidogyne incognita forms conspicuous tubular structures referred to as feeding tubes in special food cells, called giant-cells, induced and maintained in susceptible host roots by feeding nematodes. Feeding tubes are formed by nematode secretions injected into giant-cells via a stylet and apparently function to facilitate withdrawal of soluble assimilates by the parasite. In giant-cells in roots of the four host species examined in this study, feeding tube morphology was identical. Tubes were straight to slightly curved structures just less than 1 μm wide and up to slightly more than 70 μm long. At the ultrastructural level, each tube consisted of a 190–290 nm thick, electron-dense, crystalline wall surrounding an electron-transparent lumen with a diameter of 340–510 nm. The distal end of the tube was sealed with wall material. Older tubes were found free in the host cytoplasm while the proximal ends of young tubes were attached to the host cell wall via short wall ingrowths through which the nematode's stylet was inserted. An elaborate membrane system was associated with the feeding tubes and was most extensive around newly formed tubes. Contiguous to the feeding tube wall, this membrane system consisted of strands of smooth endoplasmic reticulum while rough endoplasmic reticulum predominated toward the outer margin of the membrane system. Vacuoles and mitochondria were excluded from a zone of cytoplasm surrounding feeding tubes. This zone of exclusion, as well as the membrane system noted above, tended to be less pronounced or absent around older tubes no longer being used by the nematode.     

Schistosomicidal Activity of Dihydrobenzofuran Neolignans

We have evaluated the antischistosomal activity of synthetic dihydrobenzofuran neolignans (DBNs) derived from (±)‐trans‐dehydrodicoumaric acid dimethyl ester ( 1 ) and (±)‐trans‐dehydrodiferulic acid dimethyl ester ( 2 ) against adult Schistosoma mansoni worms in vitro. Compound 4 ((±)‐trans‐4‐O‐acetyldehydrodiferulic acid dimethyl ester) displayed the most promising activity; at 200 μm , it kills 100 ± 0% of worms after 24 h, which resembles the result achieved with praziquantel (positive control) at 1.56 μm . The hydrogenation of the double bond between C7′ and C8′, the introduction of an additional methyl group at C3′, and a double bond between C7 and C8 decreased the schistosomicidal activity of DBNs. On the other hand, the presence of the acetoxy group at C4 played an interesting role in this activity. These results demonstrated the interesting schistosomicidal potential of DBNs, which could be further exploited.     

HOLDING ON BY THEIR HOOKS: ANCHORS FOR WORMS

We examined the hooked setae of a dominant group of tube-dwellers, the polychaete annelids, and found a pattern of setation that is predictable by tube type, exclusive of worm taxon or orientation; we also demonstrated the mechanical significance of these hooked setae. When tube-dwelling worms belonging to different lineages are pressurized, they resist differentially as a function of the direction in which hooks face. The results are consistent with the hypothesis that hooks are used primarily to resist removal of worms from their tubes, are polyphyletic in origin, and are active agents of resistance.     

Fast pollen tube growth in Conospermum species

BACKGROUND AND AIMS: An unusual form of pollen tube growth was observed for several Conospermum species (family Proteaceae). The rate of pollen tube growth, the number of tubes to emerge and the ultrastructure of these tubes are given here. METHODS: Pollen was germinated in vitro in different sucrose concentrations and in the presence of calcium channel blockers, and tube emergence and growth were recorded on a VCR. Measurements were taken of the number of tubes to emerge and rate of tube emergence. Pollen behaviour in vivo was also observed. The ultrastructure of germinated and ungerminated pollen was observed using TEM. RESULTS: After 10 s to 3 min in germination medium, up to three pollen tubes emerged and grew at rates of up to 55 micro m s(-1); the rate then slowed to around 2 micro m s(-1), 30 s after the initial growth spurt. Tubes were observed to grow in pulses, and the pulsed growth continued in the presence of calcium channel blockers. Optimal sugar concentration for pollen germination was 300 g L(-1), in which up to 81 % of pollen grains showed fast germination. Germination and emergence of multiple tubes were observed in sucrose concentrations of 100-800 g L(-1). The vegetative and generative nuclei moved into one of the tubes. Multiple tubes from a single grain were observed on the stigma. Under light microscopy, the cytoplasm in the tube showed a clear region at the tip. The ultrastructure of C. amoenum pollen showed a bilayered exine, with the intine being very thick at the pores, and elsewhere having large intrusions into the plasma membrane. The cytoplasm was dense with vesicles packed with inner tube cell wall material. Golgi apparatus producing secretory vesicles, and mitochondria were found throughout the tube. The tube wall was bilayered; both layers being fibrous and loosely packed. CONCLUSIONS: It is proposed that, for Conospermum, initial pollen tube wall constituents are manufactured and stored prior to pollen germination, and that tube extension occurs as described in the literature for other species, but at an exceptionally fast rate.     

Parasitic worms hijack key plant protein to build their nest

Parasitic nematode worms infect a variety of crop plants worldwide. Roots infected by these worms start to look rather unsavory – with knot like tumors (galls) developing all over them. At the core of each gall, a worm matures and lays its eggs. Olmo et al. (2018) looked into the developmental reprogramming that leads to gall formation and found an Arabidopsis protein to be a necessary component in this process.     

Microvascular anatomy and histomorphology of extrapulmonary bronchi in adult Xenopus laevis Daudin (Lissamphibia; Anura) point to a role in aerial gas exchange – histomorphology of tissue sections and scanning electron microscopy of vascular corrosion casts

Studies on the amphibian respiratory tract microvascular anatomy are few. Using scanning electron microscopy (SEM) of vascular corrosion casts (VCCs) and light microscopy of perfusion‐fixed tissue sections, we studied the bronchial microvascular anatomy in the adult South African Clawed Toad, Xenopus laevis Daudin. Histomorphology showed that the bronchial wall consists (from luminal to abluminal) of squamous epithelium, subepithelial capillary bed, cartilage rings or cartilage plates, a layer of dense connective tissue, a layer of smooth muscle cells, and squamous epithelium (serosa). SEM of VCCs reveals that bilaterally a ventral, a dorsal (Ø 77.21 ± 7.61 μm), and a caudal bronchial artery supply the bronchial subepithelial capillary bed. The ventral bronchial artery has 3–4 branching orders (interbranching distances: 506.3 ± 392.12 μm; branching angles of first‐ and second‐order bifurcations: 24.60 ± 10.24° and 29.59 ± 14.3°). Casts of bronchial arteries display imprints of flow dividers and sphincters. Cranial and caudal bronchial veins (Ø 154.78 ± 49.68 μm) drain into pulmonary veins. They lack microvenous valves. The location of the dense subepithelial capillary meshwork just beneath the thin squamous bronchial epithelium and its drainage into the pulmonary veins make it likely that in Xenopus, bronchi assist in aerial gas exchange.     

Aquatic worms eating waste sludge in a continuous system

Aquatic worms are a biological approach to decrease the amount of biological waste sludge produced at waste water treatment plants. A new reactor concept was recently introduced in which the aquatic oligochaete Lumbriculus variegatus is immobilised in a carrier material. The current paper describes the experiments that were performed to test whether this concept could also be applied in continuous operation, for which worm growth is an important condition. This was tested for two mesh sizes of the carrier material. With an increase in mesh size from 300 to 350 μm, worm biomass growth was possible in the reactor at a rate of 0.013 d⁻¹ and with a yield of 0.13 g dw/g VSS digested by the worms. Mass balances over the worm reactors showed the importance of correcting for natural sludge breakdown, as the contribution of the worms to total VSS reduction was 41–71%.     

Effects of temperature on germination and hyphal growth from ascospores of A-group and B-group Leptosphaeria maculans (phoma stem canker of oilseed rape)

Ascospores of both A‐group and B‐group Leptosphaeria maculans germinated at temperatures from 5–20°C on distilled water agar or detached oilseed rape leaves. After 2 h of incubation on water agar, some A‐group ascospores had germinated at 10–20°C and some B‐group ascospores had germinated at 5–20°C. The percentages of both A‐group and B‐group ascospores that had germinated after 24 h of incubation increased with increasing temperature from 5–20°C. The observed time (Vo₅₀) which elapsed from inoculation until 50% of the spores had germinated was shorter for B‐group than for A‐group ascospores. Germ tube length increased with increasing temperature from 5–20°C for both ascospore groups. Germ tubes from B‐group ascospores were longer than germ tubes from A‐group ascospores at all temperatures tested, but the mean diameter of germ tubes from A‐group ascospores (1.8 μm) was greater than that of those from B‐group ascospores (1.2μm) at 15°C and 20°C. The average number of germ tubes produced from A‐group ascospores (3.8) was greater than that from B‐group ascospores (3.1) after 24 h of incubation at 20°C, on both water agar and leaf surfaces. Germ tubes originated predominantly from interstitial cells or terminal cells of A‐group or B‐group ascospores, respectively, on both water agar and leaf surfaces. Hyphae from A‐group ascospores grew tortuously with extensive branching, whilst those from B‐group ascospores were predominantly long and straight with little branching, whether the ascospores were produced from oilseed rape debris or from crosses between single ascospore isolates, and whether ascospores were germinating on water agar or leaf surfaces.     

Chemical Composition,Antibacterial, Schistosomicidal,and Cytotoxic Activities of the Essential Oil of Dysphania ambrosioides (L.) Mosyakin & Clemants (Chenopodiaceae)

We have investigated the chemical composition and the antibacterial activity of the essential oil of Dysphania ambrosioides (L.) Mosyakin & Clemants (Chenopodiaceae) (DA‐EO) against a representative panel of cariogenic bacteria. We have also assessed the in vitro schistosomicidal effects of DA‐EO on Schistosoma mansoni and its cytotoxicity to GM07492‐A cells in vitro. Gas chromatography (GC) and gas chromatography‐mass spectrometry (GC/MS) revealed that the monoterpenes cis‐piperitone oxide (35.2%), p‐cymene (14.5%), isoascaridole (14.1%), and α‐terpinene (11.6%) were identified by as the major constituents of DA‐EO. DA‐EO displayed weak activity against Streptococcus sobrinus and Enterococcus faecalis (minimum inhibitory concentration (MIC) = 1000 μg/ml). On the other hand, DA‐EO at 25 and 12.5 μg/ml presented remarkable schistosomicidal action in vitro and killed 100% of adult worm pairs within 24 and 72 h, respectively. The LC₅₀ values of DA‐EO were 6.50 ± 0.38, 3.66 ± 1.06, and 3.65 ± 0.76 μg/ml at 24, 48, and 72 h, respectively. However, DA‐EO at concentrations higher than 312.5 μg/ml significantly reduced the viability of GM07492‐A cells (IC₅₀ = 207.1 ± 4.4 μg/ml). The selectivity index showed that DA‐EO was 31.8 times more toxic to the adult S. mansoni worms than GM07492‐A cells. Taken together, these results demonstrate the promising schistosomicidal potential of the essential oil of Dysphania ambrosioides.     

Kraftionema allantoideum,a new genus and family of Ulotrichales (Chlorophyta) adapted for survival in high intertidal pools

The marine, sand‐dwelling green alga Kraftionema allantoideum gen. et sp. nov. is described from clonal cultures established from samples collected in coastal, high intertidal pools from south eastern Australia. The species forms microscopic, uniseriate, unbranched, 6–8 μm wide filaments surrounded by a gelatinous capsule of varying thickness. Filaments are twisted, knotted, and variable in length from 4 to 50 cells in field samples but straighter and much longer in culture, up to 1.5 mm in length. Cell division occurs in several planes, resulting in daughter cells of varying shape, from square to rectangular to triangular, giving rise to gnarled filaments. Mature cells become allantoid, elongate with rounded ends, before dividing one time to form bicells comprised of two domed cells. Adjacent bicells separate from one another and mature filaments appeared as a string of loosely arranged sausages. A massive, single, banded chloroplast covered 3/4 of the wall circumference, and contained a single large pyrenoid encased in a starch envelope that measures 1.5–2.5 μm. Filaments were not adhesive nor did they produce specialized adhesive cells or structures. Reproduction was by fragmentation with all cells capable of producing a new filament. No motile or reproductive cells were observed. Filaments in culture grew equally well in freshwater or marine media, as well as at high salinity, and cells quickly recovered from desiccation. Phylogenetic analysis based on the nuclear‐encoded small subunit ribosomal RNA (18S) shows the early branching nature of the Kraftionema lineage among Ulotrichales, warranting its recognition as a family (Kraftionemaceae).     

The exogenous application of AtPGLR,an endo‐polygalacturonase,triggers pollen tube burst and repair

Plant cell wall remodeling plays a key role in the control of cell elongation and differentiation. In particular, fine‐tuning of the degree of methylesterification of pectins was previously reported to control developmental processes as diverse as pollen germination, pollen tube elongation, emergence of primordia or elongation of dark‐grown hypocotyls. However, how pectin degradation can modulate plant development has remained elusive. Here we report the characterization of a polygalacturonase (PG), AtPGLR, the gene for which is highly expressed at the onset of lateral root emergence in Arabidopsis. Due to gene compensation mechanisms, mutant approaches failed to determine the involvement of AtPGLR in plant growth. To overcome this issue, AtPGLR has been expressed heterologously in the yeast Pichia pastoris and biochemically characterized. We showed that AtPGLR is an endo‐PG that preferentially releases non‐methylesterified oligogalacturonides with a short degree of polymerization (< 8) at acidic pH. The application of the purified recombinant protein on Amaryllis pollen tubes, an excellent model for studying cell wall remodeling at acidic pH, induced abnormal pollen tubes or cytoplasmic leakage in the subapical dome of the pollen tube tip, where non‐methylesterified pectin epitopes are detected. Those leaks could either be repaired by new β‐glucan deposits (mostly callose) in the cell wall or promoted dramatic burst of the pollen tube. Our work presents the full biochemical characterization of an Arabidopsis PG and highlights the importance of pectin integrity in pollen tube elongation.     

PELPIII: the class III pistil‐specific extensin‐like Nicotiana tabacum proteins are essential for interspecific incompatibility

Pre‐zygotic interspecific incompatibility (II) involves an active inhibition mechanism between the pollen of one species and the pistil of another. As a barrier to fertilization, II effectively prevents hybridization and maintains species identity. Transgenic ablation of the mature transmitting tract (TT) in Nicotiana tabacum resulted in the loss of inhibition of pollen tube growth in Nicotiana obtusifolia (synonym Nicotiana trigonophylla) and Nicotiana repanda. The role of the TT in the II interaction between N. tabacum and N. obtusifolia was characterized by evaluating N. obtusifolia pollen tube growth in normal and TT‐ablated N. tabacum styles at various post‐pollination times and developmental stages. The II activity of the TT slowed and then arrested N. obtusifolia pollen tube growth, and was developmentally synchronized. We hypothesize that proteins produced by the mature TT and secreted into the extracellular matrix inhibit interspecific pollen tubes. When extracts from the mature TT of N. tabacum were injected into the TT‐ablated style prior to pollination, the growth of incompatible pollen tubes of N. obtusifolia and N. repanda was inhibited. The class III pistil‐specific extensin‐like protein (PELPIII) was consistently associated with specific inhibition of pollen tubes, and its requirement for II was confirmed through use of plants with antisense suppression of PELPIII. Inhibition of N. obtusifolia and N. repanda pollen tube growth required accumulation of PELPIII in the TT of N. tabacum, supporting PELPIII function in pre‐zygotic II.