Information

Identification of unknown bagworm (Singapore)


This bagworm was found in Singapore (near McRitchie Reservoir) some time in June 2012.

This is the plant from which the bagworms were obtained:

A few examples of the bagworms:

The bagworm when dissected out of its bag:


Is the plant Banana (looks similar)? If so, the bagworm (Psychidae) Kophene cuprea could be a possibility, since it is considered a pest on banana (Mosich & Larsen, 1978). The larval case is also supposed to be conical, but I cannot find an online picture for comparison.


DNA Testing To Identify a Birth Parent

If you have one known parent and one unknown parent, you may be able to use a pretty straightforward process to identify your unknown parent, or at least to learn more about that parent’s family. I’ll explain this process using a real scenario, with details changed for privacy reasons. But you can follow these same steps yourself, adapting them for your own situation.


Identify Anions

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The following table shows the tests for anions:
Carbonate, Chloride, Bromide, Iodide, Nitrate, Sulfate and Sulfite.

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Open Research

Data available on request due to privacy/ethical restrictions

Table S1 Table showing the trend of patient's full blood count, liver enzymes including alanine transaminase (ALT), aspartate transaminase (AST), gamma-glutamyl transferase (GGT)

Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.


Results

Identification of gametophytic mutants

With the purpose of identifying mutants of Arabidopsis thalianawith defects in female gametophyte development, we performed a large-scale insertional mutagenesis screen using an Ac/Ds transposon system(Sundaresan et al., 1995). As the Ds element includes an NPTII gene conferring kanamycin resistance, plants carrying a transposed Ds element were tested for segregation ratio distortion in kanamycin resistance as a first indicative of a gametophytic defect. As the lines carry a single Ds insertion, the progeny of plants heterozygous for the insertion should segregate for kanR:kanS at 3:1. A distortion of this 3:1 segregation ratio indicates that the transposon has disrupted a gene required either for gametophyte or embryo viability. Although ratios in the order of 2:1 are indicative of embryo lethality, a ratio lower than 2:1 suggests that the Ds insertion has affected gametophyte development or function. A ratio of 1:1 would result from lethality or failure to transmit through either the male or female gametophyte, and ratios lower than 1:1 result from significant reduction in viability of both gametophytes. Ds insertion lines(24,000) containing either a Ds gene trap or an enhancer trap element were screened for segregation distortion. A total of 333 lines (1.38%)segregating at Kan R ratios of 1.5:1 or less were identified. To determine the viability of the male and female gametophytes and the penetrance of the mutations in these lines, we crossed the heterozygous mutant lines as females using wild-type plants as sperm donors, and vice versa, and scored the number of heterozygous (kanamycin-resistant) and homozygous wild-type(kanamycin-sensitive) resulting progeny. From our analysis of 333 mutants, a total of 162 lines exhibited reduced transmission efficiency when crossed as females (Table 1). Out of the 162 female mutant lines, 28 were fully penetrant mutants (i.e. TE=0). The detailed segregation distortion data from self crosses can be found in Table S1 in the supplementary material). For this study, we selected 130 mutant lines for further characterization (see below).

Transmission efficiencies detected in potential female gametophytic mutant lines

Plants were crossed manually and seeds of the resulting cross were collected and grown on kanamycin-containing MS plates to determine the efficiency in which the mutant allele (carrying the kanamycin resistance) was transmitted to the next generation by the females and male gametes. TE was calculated as the ratio of kanamycin-resistant plants to kanamycin-sensitive plants resulting from the crossing indicated.

Phenotypic analysis of the female mutants

In the developing wild-type ovule, a single hypodermal cell of the nucellus is specified as the archesporial cell (although occasional exceptions can be observed) (Grossniklaus and Schneitz,1998). The archesporial cell enlarges and differentiates into the female meiocyte or megaspore mother cell (MMC). The MMC undergoes meiosis to generate four spores, three of which undergo programmed cell death, leaving only the proximal (chalazal) megaspore as the functional megaspore in each ovule (see Fig. 1). The megaspore then undergoes three sequential mitotic nuclear divisions, to generate the eight nuclei of the mature embryo sac. Subsequent cellularization results in the formation of just seven cells, owing to nuclear migration and eventual fusion of two nuclei in the large central cell. In the mature embryo sac, the micropylar (distal) end of the ovule has the egg cell and two supporting cells called synergids, while the chalazal (proximal) end of the ovule has three cells of undetermined function called the antipodals that disintegrate prior to fertilization (Fig. 1A). To further characterize our female mutant lines and to determine which processes of gametophyte development or function are compromised, the terminal phenotypes of 130 lines were studied by analyzing whole-mount preparation of ovules. All these mutants displayed female TE≤0.6, except for nine mutant lines that were characterized previous to obtaining the female transmission data. The mutant lines were analyzed at different phases of development, including post-pollination stages.

Wild-type female gametophyte development in Arabidopsis. (A)Scheme showing the sequential developmental events leading to the wild-type female gametophyte formation in Arabidopsis. (B) Wild-type embryo sac containing one functional megaspore. (C) Wild-type embryo sac at the two-nuclear stage. Arrowheads indicate nuclei. (D) Wild-type embryo sac at the four-nuclear stage. Arrowheads indicate nuclei. (E) Cellularized wild-type embryo sac containing eight nuclei. (F) Wild-type embryo sac containing seven cells and seven nuclei. AC, archesporial cell Ap, antipodal cells Cc,central cell Ccn, central cell nucleus Ec, egg cell FG5, eight-nuclear embryo sac FG6, seven-cell embryo sac FG7, four-cell embryo sac Fm,functional megaspore MMC, megaspore mother cell Pn, polar nuclei Syn,synergide Vac, vacuole. Scale bar: 25 μm.

Wild-type female gametophyte development in Arabidopsis. (A)Scheme showing the sequential developmental events leading to the wild-type female gametophyte formation in Arabidopsis. (B) Wild-type embryo sac containing one functional megaspore. (C) Wild-type embryo sac at the two-nuclear stage. Arrowheads indicate nuclei. (D) Wild-type embryo sac at the four-nuclear stage. Arrowheads indicate nuclei. (E) Cellularized wild-type embryo sac containing eight nuclei. (F) Wild-type embryo sac containing seven cells and seven nuclei. AC, archesporial cell Ap, antipodal cells Cc,central cell Ccn, central cell nucleus Ec, egg cell FG5, eight-nuclear embryo sac FG6, seven-cell embryo sac FG7, four-cell embryo sac Fm,functional megaspore MMC, megaspore mother cell Pn, polar nuclei Syn,synergide Vac, vacuole. Scale bar: 25 μm.

Mutants with defects in embryo sac development

As is shown in Table 2, from 130 lines, 42 (32%) exhibited obvious defects in embryo sac development, which were called EDA mutants (for embryo sac development arrest). This category includes mutants that have defects during the nuclear division phase of megagametogenesis (Fig. 2B-D),mutants presenting abnormal nuclear numbers and positions(Fig. 2E), and mutants that became cellularized, but fail in polar nuclei fusion(Fig. 2F).

Summary of phenotypes observed in insertion mutant lines with transmission deficiency through the female gametes

Phenotype observed . Number of lines .
Embryo sac with one functional megaspore 1
Embryo sac arrested at two-nuclear stage 5
Embryo sac arrested at four-nuclear stage 3
Embryo sac with varying defects in nuclei number and position 7
Unfused polar nuclei 18
Mutants showing embryo sacs arrested at varying stages of development 8
Defects in fertilization 18
Arrested at one-cell zygotic stage
Endosperm development abnormal or arrested 56
Endosperm development normal 6
Embryo development arrested 8
Total 130
Phenotype observed . Number of lines .
Embryo sac with one functional megaspore 1
Embryo sac arrested at two-nuclear stage 5
Embryo sac arrested at four-nuclear stage 3
Embryo sac with varying defects in nuclei number and position 7
Unfused polar nuclei 18
Mutants showing embryo sacs arrested at varying stages of development 8
Defects in fertilization 18
Arrested at one-cell zygotic stage
Endosperm development abnormal or arrested 56
Endosperm development normal 6
Embryo development arrested 8
Total 130

Phenotype of mutants with defects in embryo sac development. (A) Mature wild-type embryo sac. (B) Arrested embryo sac containing a functional megaspore and degenerating spores (EDA 8). (C) Mutant presenting an embryo sac arrested at the two-nuclear stage (EDA 1). The vacuole is indicated and the arrowheads indicate the nuclei. (D) Mutant with an embryo sac arrested at the four-nuclear stage (EDA 4). The vacuole is indicated and the arrowheads indicate the nuclei. (E) Embryo sac showing abnormal number and position of nuclei (EDA 13). The arrowheads indicate the nuclei positions. (F) Polar nuclei fail to fuse (EDA 27). Ant, antipodal Syn, synergide Ec, egg cellCcn, central cell nucleus Vac, vacuole Fm, functional megaspore Pn, polar nuclei. Scale bar: 25 μm.

Phenotype of mutants with defects in embryo sac development. (A) Mature wild-type embryo sac. (B) Arrested embryo sac containing a functional megaspore and degenerating spores (EDA 8). (C) Mutant presenting an embryo sac arrested at the two-nuclear stage (EDA 1). The vacuole is indicated and the arrowheads indicate the nuclei. (D) Mutant with an embryo sac arrested at the four-nuclear stage (EDA 4). The vacuole is indicated and the arrowheads indicate the nuclei. (E) Embryo sac showing abnormal number and position of nuclei (EDA 13). The arrowheads indicate the nuclei positions. (F) Polar nuclei fail to fuse (EDA 27). Ant, antipodal Syn, synergide Ec, egg cellCcn, central cell nucleus Vac, vacuole Fm, functional megaspore Pn, polar nuclei. Scale bar: 25 μm.

Within the mutants exhibiting defects during the nuclear division phase of megagametogenesis, we found a mutant arrested at stage FG1 in which the functional megaspore persisted during ovule development(Fig. 2B). We also found five mutants that progressed beyond stage FG1 but were arrested at the two-nuclear stage (Fig. 2C). All of them presented a vacuole between the two nuclei. Three mutants were found able to progress until the four-nuclear stage. Although nuclei position was normal,the mutants were arrested at this stage and the nuclei either persisted(Fig. 2D) or degenerate (not shown) during ovule development. We found another seven mutants with defects in nuclear number and positions (Fig. 2E). These mutants are characterized by presenting unusual numbers of nuclei (i.e. five nuclei, Fig. 2E), or normal number of nuclei but with aberrant distribution along the developing embryo sac (not shown). Eighteen mutants became cellularized but had defects in fusion of the polar nuclei. In this category of mutants, the polar nuclei migrated properly, but failed to fuse(Fig. 2F). Eight mutants exhibited embryo sac arrested at varying stages of megagametogenesis, showing variations within the same silique (not shown). Thus, a single silique from this class of mutant can present embryo sacs with diverse defects ranging from embryo sacs arrested at two- or four-nuclear stage, to embryo sacs with defects in polar nuclei fusion (not shown).

Mutants presenting defects in fertilization

A large fraction of mutants was found to be affected during post-pollination processes. Among these, we found 18 mutants that appeared normal at stage FG7, but were found to be unfertilized, suggesting that the mutation is affecting embryo sac functions such as pollen tube guidance or fertilization. These mutants were called UNE mutants (for unfertilized embryo sac mutants). When post-pollination stages were analyzed, some of these lines showed two intact synergids, suggesting that ovules were not attracting pollen tubes or that they failed to undergo synergid cell death(Fig. 3C).

Phenotype of mutants showing defects in fertilization and embryo development. (A) Silique showing a wild-type (Wt) and one mutant embryo (Mt)sac, both with pollen tubes (Pt) at the micropyle (UNE 10). (B) Silique showing two wild-type embryo sacs with pollen tubes at their micropyles and one mutant embryo sac that failed to attract a pollen tube to its micropyle(UNE 14). (C) Mutant embryo sac presenting two intact synergids after pollination (UNE 9). (D) Mutant showing embryo development arrested at the one-cell stage presenting two big endosperm nuclei (End n) (MEE 31). A single arrow indicates the zygote. (E) Mutant showing an abnormal embryo proper (Ep)and suspensor (MEE 70). (F) Wild-type control embryo at early heart stage. Scale bar: 25 μm. Syn, synergide Ec, egg cell Ccn, central cell nucleusF, filament.

Phenotype of mutants showing defects in fertilization and embryo development. (A) Silique showing a wild-type (Wt) and one mutant embryo (Mt)sac, both with pollen tubes (Pt) at the micropyle (UNE 10). (B) Silique showing two wild-type embryo sacs with pollen tubes at their micropyles and one mutant embryo sac that failed to attract a pollen tube to its micropyle(UNE 14). (C) Mutant embryo sac presenting two intact synergids after pollination (UNE 9). (D) Mutant showing embryo development arrested at the one-cell stage presenting two big endosperm nuclei (End n) (MEE 31). A single arrow indicates the zygote. (E) Mutant showing an abnormal embryo proper (Ep)and suspensor (MEE 70). (F) Wild-type control embryo at early heart stage. Scale bar: 25 μm. Syn, synergide Ec, egg cell Ccn, central cell nucleusF, filament.

To examine the nature of the fertilization failure further, we analyzed pollen tube growth patterns in the unfertilized mutants by Aniline Blue staining. The lines were classified according to the presence of pollen tubes at the micropyle of mutant ovules (i.e. unfertilized ovules) in comparison with the wild-type ovules present in the same pistil. When the pollen tubes were only found at the micropyle of wild-type ovules but not in the mutant ovules, the line was classified as mutant that fails to attract pollen tubes. On the one hand, the lines classified as unfertilized mutants with normal pollen tube guidance presented pollen tubes at the micropyle of at least 90%of the mutant ovules. Of 18 lines analyzed, 12 had pollen tubes at the micropyle, indicating that pollen tube guidance was not affected significantly in these cases, suggesting that failure of synergid cell death might have occurred in these mutants (Fig. 3A). On the other hand, four mutants failed to attract pollen tubes (Fig. 3B) and two mutant lines exhibited abnormal pollen tube growth patterns, which are currently under characterization. These defects in pollen tube attraction and guidance were also observed when wild-type plants were used as pollen donors (not shown), confirming that the defects arise due to disruption of female gametophytic functions.

Mutants presenting defects in embryogenesis

An unexpectedly large number of mutants, represented by 62 lines (48%),exhibited fertilized embryo sacs in which the embryo development was arrested at the one-cell zygotic stage (Table 2, Fig. 3D). In general, endosperm development was also arrested in these mutants, containing one to four big nuclei (Fig. 3D). However, six of the zygotic-arrest mutants showed normal endosperm development (not shown). To determine if these mutants were due to defects in the female gametophyte, we crossed all 62 mutant lines as females using wild-type plants as pollen donors and we examined the resulting progeny by scoring the number of mutant embryo sacs per silique at 24 to 48 hours after pollination. In all the cases, the ratio of mutant to wild-type embryo sacs found was at least 35%, demonstrating that the zygotic arrest arises from the female gametophyte and is not rescued by wild-type pollen.

Eight mutants were found arrested at later stages of embryo development,ranging from two-cell stage to globular and early heart stages(Fig. 3E). These mutants are characterized by a slight general delay in embryo sac development and fertilization (up to 12 hours), with various levels of endosperm development. When these lines were crossed as females using wild-type plants as pollen donors, the ratio of mutant to wild-type embryos in the silique was 35-50%,indicating that the embryo arrest phenotype in this class also arises from a mutation of the female gametophyte.

These mutants with defects in embryogenesis were called MEE mutants (for maternal effect embryo arrest).

Identification of the genes involved in female gametophytic mutations

Thermal asymmetric interlaced PCR (TAIL-PCR)(Liu et al., 1995) was performed to isolate the flanking sequence surrounding the Ds element using Ds-specific primers and arbitrary degenerated primers. The chromosomal location of the flanking sequences was determined by nucleotide BLAST searches and further confirmed by PCR using gene-specific primers in combination with a Ds element primer. Using the current annotation of the Arabidopsisgenome, two out of the 127 (1.5%) insertion sites sequenced showed to be within a predicted gene (hypothetical protein), while 19 (15%) are within genes encoding proteins with an EST match but without any protein match(unknown proteins). The rest of the tagged genes were classified according to their biological role or biochemical function as described by Lin et al.(Lin et al., 1999). A distribution of the genes disrupted in the female gametophytic mutants is shown in Fig. 4, while the identity of the disrupted genes classified by phenotypic category can be found in Table 3 (a fuller description of the genes based on the current annotation in the TAIR database can be found in Table S2 in the supplementary material). As unlikely, but possible, excision and nearby insertions of the Ds element could have occurred prior to the establishment of the stable insertion line, it is probable that although the mutants are tightly linked to the Ds insertion, some of the mutants are not tagged. The first class of mutant lines represents mutants with obvious defects in the development of the embryo sac. A large fraction(13%) of these mutants represented disruptions of protein degradation pathways. However, these were restricted to mutants that are arrested during the nuclear division phase of megagametogenesis or in mutants presenting embryo sacs with abnormal nuclear numbers and positions(Fig. 4A). Genes implicated in secondary metabolism, transcriptional regulation and signal transduction were also found to be involved both in the nuclear division phase and in the nuclear fusion phase. A transmembrane receptor protein was identified (line EDA 23, Table 3) as well as a guanine nucleotide exchange protein (Line EDA 10, Table 3). The latter protein is an interactor of PRK1 (Park et al.,2000), which is a receptor-like kinase with serine/threonine kinase activity isolated from petunia and involved in embryo sac development(Lee et. al, 1997). Genes encoding proteins involved in electron-transferring systems (classified as proteins involved in energy metabolism) were found disrupted in those mutants exhibiting un-fused polar nuclei. Genes for calmodulin-binding proteins and calcium-binding proteins were also found related to polar nuclei fusion (line EDA 39 and EDA 34, Table 3). Different classes of transcription factors were identified including basic-helix-loop-helix-type transcription factors and CCAAT-binding factors(Fig. 4A, Table 3).

Distribution and classification of the genes disrupted in mutants with defects in embryo sac development (A), fertilization process (B) and early stages of embryo development (C).

Distribution and classification of the genes disrupted in mutants with defects in embryo sac development (A), fertilization process (B) and early stages of embryo development (C).

Identity of the genes disrupted by Ds::KanR in female gametophytic mutants grouped by phenotypic category with the transmission ratios for each mutant gene

General defect observed . Mutant line ID . DS element location . Kan R :Kan S crossed as female . Phenotype observed .
In nuclear division phase of megagametogenesis EDA1 At1g59680 0.0092 Arrested at two-nuclear stage
EDA2 At2g18080 * 0.0000 Arrested at two-nuclear stage
EDA3 At2g34860 0.0029 Arrested at two-nuclear stage
EDA4 At2g48140 0.0000 Arrested at four-nuclear stage
EDA 5 At3g03650 0.1900 Arrested at four-nuclear stage
EDA 6 At3g23440 0.3500 Arrested at two-nuclear stage
EDA 7 At3g56990 0.0090 Arrested at four-nuclear stage
EDA 8 At4g00310 * 0.3300 Arrested at stage FG1
EDA 9 At4g34200 0.0000 Arrested at two-nuclear stage
Abnormal nuclear numbers and positions EDA 10 At1g01960 0.4700 Variant nuclear number or not embryo sac
EDA 11 At1g55420 0.1700 Variant nuclear number and positions
EDA 12 At2g35950 0.5200 Variant nuclear number and positions
EDA 13 At2g47990 0.0000 Variant nuclear number and positions
EDA 14 At3g60360 0.0000 Abnormal positions and size four-nuclear stage
EDA 15 At4g14790 0.4400 Variant nuclear number and positions
Arrested at varying stages of embryo sac development EDA 16 At1g61140 0.4600 Aberrant embryo sacs and with unfused polar nuclei
EDA 17 At1g72970 0.1800 Aberrant embryo sac and arrested at two-nuclear stage
EDA 18 At2g34920 0.3500 Aberrant embryo sacs and with unfused polar nuclei
EDA 19 At2g47990 0.0380 Aberrant embryo sacs and with unfused polar nuclei
EDA 20 At4g00020 0.2300 Aberrant embryo sacs and unfertilized ovules
EDA 21 At4g13235 0.4900 Aberrant embryo sacs and with unfused polar nuclei
EDA 22 At5g05920 0.3000 Arrested at four-nuclear stage and unfused polar nuclei
EDA 23 At5g44700 0.1974 Arrested at two-nuclear stage and unfused polar nuclei
Unfused polar nuclei EDA 24 At1g70540 0.0208 Unfused polar nuclei
EDA 25 At1g72440 0.5846 Unfused polar nuclei
EDA 26 At2g01730 0.4100 Unfused polar nuclei
EDA 27 At2g20490 0.0000 Unfused polar nuclei
EDA 28 At2g34790 0.4100 Unfused polar nuclei
EDA 29 At2g35940 0.5100 Unfused polar nuclei
EDA 30 At3g03810 0.1200 Unfused polar nuclei
EDA 31 At3g10000 0.4600 Unfused polar nuclei
EDA 32 At3g62210 0.4773 Unfused polar nuclei
EDA 33 At4g00120 0.5200 Unfused polar nuclei
EDA 34 At4g00140 0.1573 Unfused polar nuclei
EDA 35 At4g05450 0.0350 Unfused polar nuclei
EDA 36 At4g13890 0.2500 Unfused polar nuclei
EDA 37 At4g13890 0.2500 Unfused polar nuclei
EDA 38 At4g14040 0.6840 Unfused polar nuclei
EDA 39 At4g33050 0.6200 Unfused polar nuclei
EDA 40 At4g37890 0.5844 Unfused polar nuclei
EDA 41 At5g52460 0.2900 Unfused polar nuclei
Defects in fertilization UNE 1 At1g29300 0.2000 Defects in pollen tube attraction
UNE 2 At1g78130 0.2900 Unfertilized ovules but normal pollen tube attraction
UNE 3 At2g01110 0.7143 Unfertilized ovules but normal pollen tube attraction
UNE 4 At2g12940 0.3500 Defects in pollen tube attraction
UNE 5 At2g47470 0.7813 Unfertilized ovules but normal pollen tube attraction
UNE 6 At3g03340 0.8200 Unfertilized ovules but normal pollen tube attraction
UNE 7 At3g03690 0.2800 Unfertilized ovules but normal pollen tube attraction
UNE 8 At3g05690 0.1800 Unfertilized ovules but normal pollen tube attraction
UNE 9 At3g10560 0.3400 Defects in pollen tube attraction
UNE 10 At4g00050 0.4500 Unfertilized ovules but normal pollen tube attraction
UNE 11 At4g00080 0.6417 Defects in pollen tube attraction
UNE 12 At4g02590 0.5200 Unfertilized ovules but normal pollen tube attraction
UNE 13 At4g12620 0.3700 Unfertilized ovules but normal pollen tube attraction
UNE 14 At4g12860 0.5800 Defects in pollen tube attraction
UNE 15 At4g13560 0.4900 Defects in pollen tube attraction
UNE 16 At4g13640 0.2400 Unfertilized ovules but normal pollen tube attraction
UNE 17 At4g26330 0.2300 Unfertilized ovules but normal pollen tube attraction
UNE 18 At5g02100 0.1400 Unfertilized ovules but normal pollen tube attraction
Arrested at one-cell zygotic stage MEE 1 ND 0.3800 Endosperm development arrested
MEE 2 ND 0.5600 Endosperm development arrested
MEE 3 At2g21650 * 0.0610 Endosperm development arrested
MEE 4 At1g04630 0.0050 Endosperm development arrested
MEE 5 At1g06220 0.4100 Endosperm development arrested
MEE 6 At1g07890 0.0420 Endosperm development arrested
MEE 7 At1g10470 0.4400 Endosperm development arrested
MEE 8 At1g25310 0.1642 Endosperm development arrested
MEE 9 At1g60870 0.3100 Endosperm development arrested
MEE 10 At2g01200 0.5000 Endosperm development arrested
MEE 11 At2g01620 0.0000 Endosperm development arrested
MEE 12 At2g02955 0.4200 Endosperm development arrested
MEE 13 At2g14680 0.0000 Endosperm development arrested
MEE 14 At2g15890 0.0240 Endosperm development arrested
MEE 15 At2g16970 0.4300 Endosperm development arrested
MEE 16 At2g18650 0.0190 Endosperm normal
MEE 17 At2g22250 0.3333 Endosperm normal
MEE 18 At2g34090 0.2900 Endosperm development arrested
MEE 19 At2g34130 0.4800 Endosperm development arrested
MEE 20 At2g34220 0.1800 Endosperm development arrested
MEE 21 At2g34570 0.3800 Endosperm development arrested
MEE 22 At2g34780 0.4900 Endosperm development arrested
MEE 23 At2g34790 0.3913 Endosperm development arrested
MEE 24 At2g34830 0.2900 Endosperm development arrested
MEE 25 At2g34850 0.3000 Endosperm development arrested
MEE 26 At2g34870 0.4600 Endosperm development arrested
MEE 27 At2g34880 0.4800 Endosperm development arrested
MEE 28 At2g35210 0.4083 Endosperm development arrested
MEE 29 At2g35340 0.4100 Endosperm development arrested
MEE 30 At2g47470 0.2600 Endosperm development arrested
MEE 31 At3g02570 0.5300 Endosperm development arrested
MEE 32 At3g06350 0.5328 Endosperm development arrested
MEE 33 At3g10920 0.0556 Endosperm normal
MEE 34 At3g11270 0.2353 Endosperm normal
MEE 35 At3g15030 0.0837 Endosperm development arrested
MEE 36 At3g16440 0.0000 Endosperm development arrested
MEE 37 At3g23440 0.3000 Endosperm development arrested
MEE 38 At3g43160 0.4800 Endosperm development arrested
MEE 39 At3g46330 0.4700 Endosperm development arrested
MEE 40 At3g53700 0.4300 Endosperm development arrested
MEE 41 At3g62670 0.1800 Endosperm development arrested
MEE 42 At3g63080 0.5900 Endosperm development arrested
MEE 43 At4g00020 0.5100 Endosperm normal
MEE 44 At4g00060 0.2500 Endosperm development arrested
MEE 45 At4g00260 0.3519 Endosperm development arrested
MEE 46 At4g00310 0.2800 Endosperm development arrested
MEE 47 At4g00950 0.3400 Endosperm development arrested
MEE 48 At4g14080 * 0.2200 Endosperm development arrested
MEE 49 At4g01560 0.1900 Endosperm development arrested
MEE 50 At4g00231 0.5700 Endosperm development arrested
MEE 51 At4g04040 0.1000 Endosperm development arrested
MEE 52 At4g04160 0.0435 Endosperm development arrested
MEE 53 At4g10560 0.1100 Endosperm normal
MEE 54 At4g11850 0.5500 Endosperm development arrested
MEE 55 At4g13345 0.4600 Endosperm development arrested
MEE 56 At4g13380 0.4300 Endosperm development arrested
MEE 57 At4g13610 0.5800 Endosperm development arrested
MEE 58 At4g13940 0.6593 Endosperm development arrested
MEE 59 At4g37300 0.3093 Endosperm development arrested
MEE 60 At5g05950 0.2500 Endosperm development arrested
MEE 61 At5g14220 0.4000 Endosperm development arrested
MEE 62 At5g45800 0.7714 Endosperm development arrested
Embryo development defects MEE 63 At1g02140 0.8300 Embryos arrested at various stages of development
MEE 64 At1g79860 0.4500 Arrested at the two-cell embryo stage
MEE 65 At2g01280 0.6852 Arrested at early embryo stages
MEE 66 At2g02240 0.2000 Arrested at the two-cell embryo stage
MEE 67 At3g10110 0.3400 Arrested at the two-cell embryo stage
MEE 68 At4g24660 0.0972 Arrested at early embryo stages
MEE 69 At4g37140 0.2300 Arrested at the two-cell embryo stage
MEE 70 At5g58230 0.0000 Abnormal embryo development
General defect observed . Mutant line ID . DS element location . Kan R :Kan S crossed as female . Phenotype observed .
In nuclear division phase of megagametogenesis EDA1 At1g59680 0.0092 Arrested at two-nuclear stage
EDA2 At2g18080 * 0.0000 Arrested at two-nuclear stage
EDA3 At2g34860 0.0029 Arrested at two-nuclear stage
EDA4 At2g48140 0.0000 Arrested at four-nuclear stage
EDA 5 At3g03650 0.1900 Arrested at four-nuclear stage
EDA 6 At3g23440 0.3500 Arrested at two-nuclear stage
EDA 7 At3g56990 0.0090 Arrested at four-nuclear stage
EDA 8 At4g00310 * 0.3300 Arrested at stage FG1
EDA 9 At4g34200 0.0000 Arrested at two-nuclear stage
Abnormal nuclear numbers and positions EDA 10 At1g01960 0.4700 Variant nuclear number or not embryo sac
EDA 11 At1g55420 0.1700 Variant nuclear number and positions
EDA 12 At2g35950 0.5200 Variant nuclear number and positions
EDA 13 At2g47990 0.0000 Variant nuclear number and positions
EDA 14 At3g60360 0.0000 Abnormal positions and size four-nuclear stage
EDA 15 At4g14790 0.4400 Variant nuclear number and positions
Arrested at varying stages of embryo sac development EDA 16 At1g61140 0.4600 Aberrant embryo sacs and with unfused polar nuclei
EDA 17 At1g72970 0.1800 Aberrant embryo sac and arrested at two-nuclear stage
EDA 18 At2g34920 0.3500 Aberrant embryo sacs and with unfused polar nuclei
EDA 19 At2g47990 0.0380 Aberrant embryo sacs and with unfused polar nuclei
EDA 20 At4g00020 0.2300 Aberrant embryo sacs and unfertilized ovules
EDA 21 At4g13235 0.4900 Aberrant embryo sacs and with unfused polar nuclei
EDA 22 At5g05920 0.3000 Arrested at four-nuclear stage and unfused polar nuclei
EDA 23 At5g44700 0.1974 Arrested at two-nuclear stage and unfused polar nuclei
Unfused polar nuclei EDA 24 At1g70540 0.0208 Unfused polar nuclei
EDA 25 At1g72440 0.5846 Unfused polar nuclei
EDA 26 At2g01730 0.4100 Unfused polar nuclei
EDA 27 At2g20490 0.0000 Unfused polar nuclei
EDA 28 At2g34790 0.4100 Unfused polar nuclei
EDA 29 At2g35940 0.5100 Unfused polar nuclei
EDA 30 At3g03810 0.1200 Unfused polar nuclei
EDA 31 At3g10000 0.4600 Unfused polar nuclei
EDA 32 At3g62210 0.4773 Unfused polar nuclei
EDA 33 At4g00120 0.5200 Unfused polar nuclei
EDA 34 At4g00140 0.1573 Unfused polar nuclei
EDA 35 At4g05450 0.0350 Unfused polar nuclei
EDA 36 At4g13890 0.2500 Unfused polar nuclei
EDA 37 At4g13890 0.2500 Unfused polar nuclei
EDA 38 At4g14040 0.6840 Unfused polar nuclei
EDA 39 At4g33050 0.6200 Unfused polar nuclei
EDA 40 At4g37890 0.5844 Unfused polar nuclei
EDA 41 At5g52460 0.2900 Unfused polar nuclei
Defects in fertilization UNE 1 At1g29300 0.2000 Defects in pollen tube attraction
UNE 2 At1g78130 0.2900 Unfertilized ovules but normal pollen tube attraction
UNE 3 At2g01110 0.7143 Unfertilized ovules but normal pollen tube attraction
UNE 4 At2g12940 0.3500 Defects in pollen tube attraction
UNE 5 At2g47470 0.7813 Unfertilized ovules but normal pollen tube attraction
UNE 6 At3g03340 0.8200 Unfertilized ovules but normal pollen tube attraction
UNE 7 At3g03690 0.2800 Unfertilized ovules but normal pollen tube attraction
UNE 8 At3g05690 0.1800 Unfertilized ovules but normal pollen tube attraction
UNE 9 At3g10560 0.3400 Defects in pollen tube attraction
UNE 10 At4g00050 0.4500 Unfertilized ovules but normal pollen tube attraction
UNE 11 At4g00080 0.6417 Defects in pollen tube attraction
UNE 12 At4g02590 0.5200 Unfertilized ovules but normal pollen tube attraction
UNE 13 At4g12620 0.3700 Unfertilized ovules but normal pollen tube attraction
UNE 14 At4g12860 0.5800 Defects in pollen tube attraction
UNE 15 At4g13560 0.4900 Defects in pollen tube attraction
UNE 16 At4g13640 0.2400 Unfertilized ovules but normal pollen tube attraction
UNE 17 At4g26330 0.2300 Unfertilized ovules but normal pollen tube attraction
UNE 18 At5g02100 0.1400 Unfertilized ovules but normal pollen tube attraction
Arrested at one-cell zygotic stage MEE 1 ND 0.3800 Endosperm development arrested
MEE 2 ND 0.5600 Endosperm development arrested
MEE 3 At2g21650 * 0.0610 Endosperm development arrested
MEE 4 At1g04630 0.0050 Endosperm development arrested
MEE 5 At1g06220 0.4100 Endosperm development arrested
MEE 6 At1g07890 0.0420 Endosperm development arrested
MEE 7 At1g10470 0.4400 Endosperm development arrested
MEE 8 At1g25310 0.1642 Endosperm development arrested
MEE 9 At1g60870 0.3100 Endosperm development arrested
MEE 10 At2g01200 0.5000 Endosperm development arrested
MEE 11 At2g01620 0.0000 Endosperm development arrested
MEE 12 At2g02955 0.4200 Endosperm development arrested
MEE 13 At2g14680 0.0000 Endosperm development arrested
MEE 14 At2g15890 0.0240 Endosperm development arrested
MEE 15 At2g16970 0.4300 Endosperm development arrested
MEE 16 At2g18650 0.0190 Endosperm normal
MEE 17 At2g22250 0.3333 Endosperm normal
MEE 18 At2g34090 0.2900 Endosperm development arrested
MEE 19 At2g34130 0.4800 Endosperm development arrested
MEE 20 At2g34220 0.1800 Endosperm development arrested
MEE 21 At2g34570 0.3800 Endosperm development arrested
MEE 22 At2g34780 0.4900 Endosperm development arrested
MEE 23 At2g34790 0.3913 Endosperm development arrested
MEE 24 At2g34830 0.2900 Endosperm development arrested
MEE 25 At2g34850 0.3000 Endosperm development arrested
MEE 26 At2g34870 0.4600 Endosperm development arrested
MEE 27 At2g34880 0.4800 Endosperm development arrested
MEE 28 At2g35210 0.4083 Endosperm development arrested
MEE 29 At2g35340 0.4100 Endosperm development arrested
MEE 30 At2g47470 0.2600 Endosperm development arrested
MEE 31 At3g02570 0.5300 Endosperm development arrested
MEE 32 At3g06350 0.5328 Endosperm development arrested
MEE 33 At3g10920 0.0556 Endosperm normal
MEE 34 At3g11270 0.2353 Endosperm normal
MEE 35 At3g15030 0.0837 Endosperm development arrested
MEE 36 At3g16440 0.0000 Endosperm development arrested
MEE 37 At3g23440 0.3000 Endosperm development arrested
MEE 38 At3g43160 0.4800 Endosperm development arrested
MEE 39 At3g46330 0.4700 Endosperm development arrested
MEE 40 At3g53700 0.4300 Endosperm development arrested
MEE 41 At3g62670 0.1800 Endosperm development arrested
MEE 42 At3g63080 0.5900 Endosperm development arrested
MEE 43 At4g00020 0.5100 Endosperm normal
MEE 44 At4g00060 0.2500 Endosperm development arrested
MEE 45 At4g00260 0.3519 Endosperm development arrested
MEE 46 At4g00310 0.2800 Endosperm development arrested
MEE 47 At4g00950 0.3400 Endosperm development arrested
MEE 48 At4g14080 * 0.2200 Endosperm development arrested
MEE 49 At4g01560 0.1900 Endosperm development arrested
MEE 50 At4g00231 0.5700 Endosperm development arrested
MEE 51 At4g04040 0.1000 Endosperm development arrested
MEE 52 At4g04160 0.0435 Endosperm development arrested
MEE 53 At4g10560 0.1100 Endosperm normal
MEE 54 At4g11850 0.5500 Endosperm development arrested
MEE 55 At4g13345 0.4600 Endosperm development arrested
MEE 56 At4g13380 0.4300 Endosperm development arrested
MEE 57 At4g13610 0.5800 Endosperm development arrested
MEE 58 At4g13940 0.6593 Endosperm development arrested
MEE 59 At4g37300 0.3093 Endosperm development arrested
MEE 60 At5g05950 0.2500 Endosperm development arrested
MEE 61 At5g14220 0.4000 Endosperm development arrested
MEE 62 At5g45800 0.7714 Endosperm development arrested
Embryo development defects MEE 63 At1g02140 0.8300 Embryos arrested at various stages of development
MEE 64 At1g79860 0.4500 Arrested at the two-cell embryo stage
MEE 65 At2g01280 0.6852 Arrested at early embryo stages
MEE 66 At2g02240 0.2000 Arrested at the two-cell embryo stage
MEE 67 At3g10110 0.3400 Arrested at the two-cell embryo stage
MEE 68 At4g24660 0.0972 Arrested at early embryo stages
MEE 69 At4g37140 0.2300 Arrested at the two-cell embryo stage
MEE 70 At5g58230 0.0000 Abnormal embryo development

Indicates sequences identified by TAIL-PCR but not confirmed yet by gene-specific primers.

A diverse set of genes were also found to be essential for fertilization,with a moderate prevalence of transcriptional regulators and proteins involved in energy metabolism (13%, Fig. 4B). Among these, proteins involved in cytochome c maturity (line MEE 30, Table 3) and with cytochrome P450 (line UNE 9, Table 3) were observed. A gene encoding an oxysterol binding protein,which is involved in oxysterol-directed apoptosis in animals(Christ et al., 1993 Schroepfer, 2000) was also detected (line UNE 18, Table 3). On the other hand, a gene encoding for an antioxidant enzyme was found (MEE 42, Table 3). For the class of mutants that showed embryo development arrested at the one-cell zygotic stage, a large and diverse collection of genes was found. A high percentage of the genes was found to encode proteins with unknown functions (20%, Fig. 4C). However, among all the genes that appear to be essential for post-zygotic development, transcriptional regulators appear to be predominant (19%, Fig. 4C). Transcription factors belonging to different families such as MYB family, WRKY family and TCP family were found. Proteins involved in signal transduction pathways also appear to be crucial for post-zygotic development. Among them, we could find receptor-like protein kinases (line MEE 39, Table 3), a phospholipase D(line MEE 54, Table 3) and a response regulator of the two-components signal transduction pathways (line MEE 7, Table 3). A gene encoding an enzyme from the porphyrin pathway was also found disrupted in one of these mutants with embryo development defects (line MEE 61, Table 3), consistent with a recent report showing that most of the enzymes from the porphyrin pathway are continuously expressed through seed development (Henning et al., 2004).

In the case of seven genes (At2g34790, At2g47470, At2g47990, At3g23440,At4g00020, At4g00310 and At4g13890), we found two independent insertion lines per gene, with the Ds element insertions at two different positions within the gene. Although, in general, similar or overlapping phenotypes were obtained when the terminal phenotype of each pair of mutant lines was compared, in the case of At3g23440 and At4g00020, the phenotype of the mutants was very different (Table 3). These divergences might arise from differences in the insertion sites of the Ds element into these genes, resulting in alleles that are not nulls but have different levels of residual expression. In the case of genes whose expression is required early for normal embryo sac development, as well as later for progression of embryogenesis, the observed differences in the terminal phenotypes might reflect such allelic variation. For example, two insertion mutants were recovered for the gene At3g23440: EDA 7 and MEE 37. Although for EDA 7 the insertion site is localized in the intergenic region downstream of the 3′ UTR, in MEE 37 the Ds element is localized in the coding region of the gene.


Computational approach to optimise culture conditions required for cell therapy

Collaboration by researchers in Singapore and Australia lead to first-of-its-kind computational biology algorithm that could enable more effective cellular therapies against major diseases.

IMAGE: The scientists used EpiMogrify, an innovative computational biology algorithm, to predict molecules needed to control the cell state and fate of cardiac muscle cells (left) and astrocytes (right). view more

Credit: Joseph Chen, Monash University

SINGAPORE / MELBOURNE -- Cellular therapy is a powerful strategy to produce patient-specific, personalised cells to treat many diseases, including heart disease and neurological disorders. But a major challenge for cell therapy applications is keeping cells alive and well in the lab.

That may soon change as researchers at Duke-NUS Medical School, Singapore, and Monash University, Australia have devised an algorithm that can predict what molecules are needed to keep cells healthy in laboratory cultures. They developed a computational approach called EpiMogrify, that can predict the molecules needed to signal stem cells to change into specific tissue cells, which can help accelerate treatments that require growing patient cells in the lab.

"Computational biology is rapidly becoming a key enabler in cell therapy, providing a way to short-circuit otherwise expensive and time-consuming discovery approaches with cleverly designed algorithms," said Assistant Professor Owen Rackham, a computational biologist at Duke-NUS, and a senior and corresponding author of the study, published today in the journal Cell Systems.

In the laboratory, cells are often grown and maintained in cell cultures, formed of a substance, called a medium, which contains nutrients and other molecules. It has been an ongoing challenge to identify the necessary molecules to maintain high-quality cells in culture, as well as finding molecules that can induce stem cells to convert to other cell types.

The research team developed a computer model called EpiMogrify that successfully identified molecules to add to cell culture media to maintain healthy nerve cells, called astrocytes, and heart cells, called cardiomyocytes. They also used their model to successfully predict molecules that trigger stem cells to turn into astrocytes and cardiomyocytes.

"Research at Duke-NUS is paving the road for cell therapies and regenerative medicine to enter the clinic in Singapore and worldwide this study leverages our expertise in computational and systems biology to facilitate the good manufacturing practice (GMP) production of high-quality cells for these much needed therapeutic applications," said Associate Professor Enrico Petretto, who leads the Systems Genetics group at Duke-NUS, and is a senior and corresponding author of the study.

The researchers added existing information into their model about genes tagged with epigenetic markers whose presence indicates that a gene is important for cell identity. The model then determines which of these genes actually code for proteins necessary for a cell's identity. Additionally, the model incorporates data about proteins that bind to cell receptors to influence their activities. Together, this information is used by the computer model to predict specific proteins that will influence different cells' identities.

"This approach facilitates the identification of the optimum cell culture conditions for converting cells and also for growing the high-quality cells required for cell therapy applications," said ARC Future Fellow Professor Jose Polo, from Monash University's Biomedicine Discovery Institute and the Australian Research Medicine Institute, who is also a senior and corresponding author of the study.

The team compared cultures using protein molecules predicted by EpiMogrify to a type of commonly used cell culture that uses a large amount of unknown or undefined complex molecules and chemicals. They found the EpiMogrify-predicted cultures worked as well or even surpassed their effectiveness.

The researchers have filed for a patent on their computational approach and the cell culture factors it predicted for maintaining and controlling cell fate. EpiMogrify's predicted molecules are available for other researchers to explore on a public database: http://epimogrify. ddnetbio. com.

"We aim to continue to develop tools and technologies that can enable cell therapies and bring them to the clinic as efficiently and safely as possible," said Asst Prof Rackham.

"The developed technology can identify cell culture conditions required to manipulate cell fate and this facilitates growing important cells in chemically-defined cultures for cell therapy applications," added Dr Uma S. Kamaraj, lead author of the study and a graduate of Duke-NUS' Integrated Biology and Medicine PhD Programme.

Disclaimer: AAAS and EurekAlert! are not responsible for the accuracy of news releases posted to EurekAlert! by contributing institutions or for the use of any information through the EurekAlert system.


Field Biology in Southeastern Ohio

That's a caterpillar? Welcome to what I find to be the most interesting moth family of all, the stinging Slug Moths, Limacodidae. Moth caterpillars have a set of prolegs under the body, these have none. Notice the smooth look to the bottom of this larva. Picture yourself with your hands tied behind your back, and you have to move across the floor on your belly. That's kind of how they get around. Because they are slow moving, these caterpillars are equipped with stinging spines. The Saddleback is one most people are familiar with. This one is Parasa chloris, the Small Green Parasa. If you would like to see a post on more of the caterpillars, click here

Here is the adult of the caterpillar above. It is one of the more colorful members of the family, and besides, there aren't a lot of green colored moths to begin with.

There are two green members of the family. The other is larger and has more green on the wings. Slug moths often sit with their wings folded down, and their butt sticking up.

The Large Parasa or Stinging Rose Caterpillar, Parasa indetemina. A slightly bigger species than the above, and with a broader green patch on the wing. The hind wings are lighter colored when compared to chloris. Diane Brooks photo.

Here is one of the VERY small species, Isa textula. The moth is light brown, and the wings have a darker rufus color.

In these photos you can see the other distinguishing marks. Rows of gray crinkly scales that stick up on the wing.

Here is a closeup of the stinging spines of Isa textula. The common name is the Crowned Slug, and you can see it appears to have a crown of thorns all around it.

Lithacodes fasciola, the Yellow-shouldered Slug. Look for the white lightning bolt in the wings for identification.

Early Button Slug, Tortricidia testacea. D. Brooks

One of the smallest species in Ohio is the Red-crossed Button Slug, Tortricidia pallida. Look for two thin lines that converge at the top of the wing. Are you starting to see the trend of wings down, butt up?

Looking like a different species, this is actually a dark form of pallida. The thin lines turn into dark blotches. At one time these were split into two species, the other being T. flexuosa. They are virtually identical, and can not even be separated by genitalia, so they are currently combined as pallida.

D. Brooks photos. As I mentioned with the previous species, the brown moth was once separated as a different species P. albipunctata, but is now simply considered a dark form of geminata.

The most common species in Ohio is the Spiny Oak Slug, Euclea delphinii. The amount of green and orange varies from specimen to specimen. Sometimes they'll have huge green patches covering the wing.

Here is a picture of the Spiny Oak Slug caterpillar. Don't sit on this guy whatever you do! It's a real whoopee-cushion. There are 18 species of Slug Moths in Ohio. This was on a Cottonwood, but it was not feeding. They feed on as many as 50 different trees and shrubs. The one plant most of them seek out is Oak. Considering we were in an oak-hickory forest, it's no surprise most of these species were found at the same time. The following are just more examples of their diversity.

Apoda biguttata, the Shagreened Slug

Yellow-collared Slug, Apoda y-inversum. D. Brooks

Adoneta bicaudata, Crested Slug

Purple-crested Slug, Adoneta spinuloides. This has the same black and white markings as the Crested Slug above, but the wing is straw colored on that species. The Purple-crested is a chocolate brown, and more commonly encountered. D. Brooks photo

The Saddleback, Acharia stimulea

This is perhaps the worst photo I took all night. I was in an extreme hurry to get this before it flew away. I was very surprised to see it. It's Packardia elegans, the Elegant Tailed Slug. It's known in Ohio from only seven specimens, all but one in northern Ohio. This is only the second time it's been recorded in the southern part of the state. Again, the photo is terrible, but a county record is a county record, and that's what's important.

Here's a much better updated pic from Diane.

Hag Moth, Phobetron pithecium. D. Brooks photos

The Slug Moths are part of the superfamily Zygaenoidea, which includes the Planthopper Parasite Moth, the Smoky Moths, and these guys, Flannel Moths. This is the Yellow Flannel Moth, Megalopyge crispata. Other common names include the Crinkled Flannel or Black-waved Flannel.

Flannel Moths get their name from the rows of raised crinkly scales on the wing that reminded someone of wool. Here's a female above and male below. The markings vary somewhat between sexes, but the antennae are distinctly different.

Here is a picture of the caterpillar. It's sometimes also called the Puss Caterpillar because the soft fur resembles that of a cat.

Make no mistake though, up close you can see under those soft hairs are a series of nasty stinging spines.

White Flannel Moth, Norape ovina.

Orange-patched Smoky Moth, Pyromorpha dimidiata. This is one of three Smoky Moths in Ohio, all of which can be found flying during the day. The wings on this species are half black and half yellow-orange.

Grapeleaf Skeletonizer. Harrisina americana. Similar to the Yellow-collared Scape Moth (Cisseps fulvicollis), this species has a brighter orange collar, a black body, and large tufts of hair at the end of the abdomen. They often hold their wings open like this when at rest. Larvae feed in groups, eating the soft tissue of grape, and leaving the main veins behind.

Our smallest species could be mistaken for a Net-winged Beetle. It's Clemens' Smoky Moth, Acoloithus falsarius. It lacks the hair tufts on the abdomen tip. The orange collar does not completely cover the neck. It is broken in the middle by black. This species keeps its wings folded roof like over the back.

Planthopper Parasite Moth, Fulgoraecia exigua. This is the sole member in Ohio of a family whose larvae feed externally on the body fluids of Homopteran plant-hoppers. Most of the specimens will show a small pale circle in the wing. This helps to separate them from the similar looking black Bagworm moths.


YhcB coordinates peptidoglycan and LPS biogenesis with phospholipid synthesis during Escherichia coli cell growth

The cell envelope is essential for viability in all kingdoms of life. It retains enzymes and substrates within a confined space while providing a protective barrier to the external environment. Destabilising the envelope of bacterial pathogens is a common strategy employed by antimicrobial treatment. However, even in one of the most well studied organisms, Escherichia coli, there remain gaps in our understanding of how the synthesis of the successive layers of the cell envelope are coordinated during growth and cell division. Here, we used a whole genome phenotypic screen to identify mutants with a defective cell envelope. We report that loss of yhcB, a conserved gene of unknown function, results in loss of envelope stability, increased cell permeability and dysregulated control of cell size. Using whole genome transposon mutagenesis strategies we report the complete genetic interaction network of yhcB, revealing all genes with a synthetic negative and a synthetic positive relationship. These genes include those previously reported to have a role in cell envelope biogenesis. Surprisingly, we identified genes previously annotated as essential that became non-essential in a ΔyhcB background. Subsequent analyses suggest that YhcB sits at the junction of several envelope biosynthetic pathways coordinating the spatiotemporal growth of the cell, highlighting YhcB as an as yet unexplored antimicrobial target.


Contents

Amoebae do not have cell walls, which allows for free movement. Amoebae move and feed by using pseudopods, which are bulges of cytoplasm formed by the coordinated action of actin microfilaments pushing out the plasma membrane that surrounds the cell. [13] The appearance and internal structure of pseudopods are used to distinguish groups of amoebae from one another. Amoebozoan species, such as those in the genus Amoeba, typically have bulbous (lobose) pseudopods, rounded at the ends and roughly tubular in cross-section. Cercozoan amoeboids, such as Euglypha and Gromia, have slender, thread-like (filose) pseudopods. Foraminifera emit fine, branching pseudopods that merge with one another to form net-like (reticulose) structures. Some groups, such as the Radiolaria and Heliozoa, have stiff, needle-like, radiating axopodia (actinopoda) supported from within by bundles of microtubules. [3] [14]

Free-living amoebae may be "testate" (enclosed within a hard shell), or "naked" (also known as gymnamoebae, lacking any hard covering). The shells of testate amoebae may be composed of various substances, including calcium, silica, chitin, or agglutinations of found materials like small grains of sand and the frustules of diatoms. [15]

To regulate osmotic pressure, most freshwater amoebae have a contractile vacuole which expels excess water from the cell. [16] This organelle is necessary because freshwater has a lower concentration of solutes (such as salt) than the amoeba's own internal fluids (cytosol). Because the surrounding water is hypotonic with respect to the contents of the cell, water is transferred across the amoeba's cell membrane by osmosis. Without a contractile vacuole, the cell would fill with excess water and, eventually, burst. Marine amoebae do not usually possess a contractile vacuole because the concentration of solutes within the cell are in balance with the tonicity of the surrounding water. [17]

The food sources of amoebae vary. Some amoebae are predatory and live by consuming bacteria and other protists. Some are detritivores and eat dead organic material.

Amoebae typically ingest their food by phagocytosis, extending pseudopods to encircle and engulf live prey or particles of scavenged material. Amoeboid cells do not have a mouth or cytostome, and there is no fixed place on the cell at which phagocytosis normally occurs. [18]

Some amoebae also feed by pinocytosis, imbibing dissolved nutrients through vesicles formed within the cell membrane. [19]

The size of amoeboid cells and species is extremely variable. The marine amoeboid Massisteria voersi is just 2.3 to 3 micrometres in diameter, [20] within the size range of many bacteria. [21] At the other extreme, the shells of deep-sea xenophyophores can attain 20 cm in diameter. [22] Most of the free-living freshwater amoebae commonly found in pond water, ditches, and lakes are microscopic, but some species, such as the so-called "giant amoebae" Pelomyxa palustris and Chaos carolinense, can be large enough to see with the naked eye.

Species or cell type Size in micrometers
Massisteria voersi [20] 2.3–3
Naegleria fowleri [23] 8–15
Neutrophil (white blood cell) [24] 12–15
Acanthamoeba [25] 12–40
Entamoeba histolytica [26] 15–60
Arcella vulgaris [27] 30–152
Amoeba proteus [28] 220–760
Chaos carolinense [29] 700–2000
Pelomyxa palustris [30] up to 5000
Syringammina fragilissima [22] up to 200 000

Some multicellular organisms have amoeboid cells only in certain phases of life, or use amoeboid movements for specialized functions. In the immune system of humans and other animals, amoeboid white blood cells pursue invading organisms, such as bacteria and pathogenic protists, and engulf them by phagocytosis. [31]

Amoeboid stages also occur in the multicellular fungus-like protists, the so-called slime moulds. Both the plasmodial slime moulds, currently classified in the class Myxogastria, and the cellular slime moulds of the groups Acrasida and Dictyosteliida, live as amoebae during their feeding stage. The amoeboid cells of the former combine to form a giant multinucleate organism, [32] while the cells of the latter live separately until food runs out, at which time the amoebae aggregate to form a multicellular migrating "slug" which functions as a single organism. [8]

Other organisms may also present amoeboid cells during certain life-cycle stages, e.g., the gametes of some green algae (Zygnematophyceae) [33] and pennate diatoms, [34] the spores (or dispersal phases) of some Mesomycetozoea, [35] [36] and the sporoplasm stage of Myxozoa and of Ascetosporea. [37]

Early history and origins of Sarcodina Edit

The earliest record of an amoeboid organism was produced in 1755 by August Johann Rösel von Rosenhof, who named his discovery "Der Kleine Proteus" ("the Little Proteus"). [38] Rösel's illustrations show an unidentifiable freshwater amoeba, similar in appearance to the common species now known as Amoeba proteus. [39] The term "Proteus animalcule" remained in use throughout the 18th and 19th centuries, as an informal name for any large, free-living amoeboid. [40]

In 1822, the genus Amiba (from the Greek ἀμοιβή amoibe, meaning "change") was erected by the French naturalist Bory de Saint-Vincent. [41] [42] Bory's contemporary, C. G. Ehrenberg, adopted the genus in his own classification of microscopic creatures, but changed the spelling to Amoeba. [43]

In 1841, Félix Dujardin coined the term "sarcode" (from Greek σάρξ sarx, "flesh," and εἶδος eidos, "form") for the "thick, glutinous, homogenous substance" which fills protozoan cell bodies. [44] Although the term originally referred to the protoplasm of any protozoan, it soon came to be used in a restricted sense to designate the gelatinous contents of amoeboid cells. [10] Thirty years later, the Austrian zoologist Ludwig Karl Schmarda used "sarcode" as the conceptual basis for his division Sarcodea, a phylum-level group made up of "unstable, changeable" organisms with bodies largely composed of "sarcode". [45] Later workers, including the influential taxonomist Otto Bütschli, emended this group to create the class Sarcodina, [46] a taxon that remained in wide use throughout most of the 20th century.

Within the traditional Sarcodina, amoebae were generally divided into morphological categories, on the basis of the form and structure of their pseudopods. Amoebae with pseudopods supported by regular arrays of microtubules (such as the freshwater Heliozoa and marine Radiolaria) were classified as Actinopoda whereas those with unsupported pseudopods were classified as Rhizopoda. [47] The Rhizopods were further subdivided into lobose, filose, and reticulose amoebae, according to the morphology of their pseudopods.

Dismantling of Sarcodina Edit

In the final decade of the 20th century, a series of molecular phylogenetic analyses confirmed that Sarcodina was not a monophyletic group. In view of these findings, the old scheme was abandoned and the amoebae of Sarcodina were dispersed among many other high-level taxonomic groups. Today, the majority of traditional sarcodines are placed in two eukaryote supergroups: Amoebozoa and Rhizaria. The rest have been distributed among the excavates, opisthokonts, and stramenopiles. Some, like the Centrohelida, have yet to be placed in any supergroup. [10] [48]

Classification Edit

Recent classification places the various amoeboid genera in the following groups:

  • Lobosa:
    • Acanthamoeba, Amoeba, Balamuthia, Chaos, Clydonella, Discamoeba, Echinamoeba, Filamoeba, Flabellula, Gephyramoeba, Glaeseria, Hartmannella, Hollandella, Hydramoeba, Korotnevella (Dactylamoeba), Leptomyxa, Lingulamoeba, Mastigina, Mayorella, Metachaos, Neoparamoeba, Paramoeba, Polychaos, Phreatamoeba, Platyamoeba, Protoacanthamoeba, Rhizamoeba, Saccamoeba, Sappinia, Stereomyxa, Thecamoeba, Trichamoeba, Trichosphaerium, Unda, Vannella, Vexillifera
    • Lobose pseudopods (Lobosa) are blunt, and there may be one or several on a cell, which is usually divided into a layer of clear ectoplasm surrounding more granular endoplasm.
      :
      • Filosa:
        • Monadofilosa: Gyromitus, Paulinella
        • Granofilosea
          : orders Aconchulinida, Pseudosporida, Reticulosida
        • Filose pseudopods (Filosa) are narrow and tapering. The vast majority of filose amoebae, including all those that produce shells, are placed within the Cercozoa together with various flagellates that tend to have amoeboid forms. The naked filose amoebae also includes vampyrellids.
        • Reticulose pseudopods (Endomyxa) are cytoplasmic strands that branch and merge to form a net. They are found most notably among the Foraminifera, a large group of marine protists that generally produce multi-chambered shells. There are only a few sorts of naked reticulose amoebae, notably the gymnophryids, and their relationships are not certain. are a subgroup of actinopods that are now grouped with rhizarians.
        • Heterolobosea:
            : Monopylocystis, Naegleria, Neovahlkampfia, Paratetramitus, Paravahlkampfia, Protonaegleria, Psalteriomonas, Sawyeria, Tetramitus, Vahlkampfia, Willaertia : Gruberella, Stachyamoeba
          • The Heterolobosea, includes protists that can transform between amoeboid and flagellate forms.
            : Chrysamoeba, Rhizochrysis : Rhizochloris
    • The heterokont chrysophyte and xanthophyte algae include some amoeboid members, the latter being poorly studied. [50]
      : Oodinium, Pfiesteria
    • Parasite with amoeboid life cycle stages.
    • Nucleariida: Micronuclearia, Nuclearia
      appear to be close relatives of animals and fungi.
    • Adelphamoeba, Astramoeba, Dinamoeba, Flagellipodium, Flamella, Gibbodiscus, Gocevia, Malamoeba, Nollandia, Oscillosignum, Paragocevia, Parvamoeba, Pernina, Pontifex, Pseudomastigamoeba, Rugipes, Striamoeba, Striolatus, Subulamoeba, Theratromyxa, Trienamoeba, Trimastigamoeba, and over 40 other genera [51]

    Some of the amoeboid groups cited (e.g., part of chrysophytes, part of xanthophytes, chlorarachniophytes) were not traditionally included in Sarcodina, being classified as algae or flagellated protozoa.

    Some amoebae can infect other organisms pathogenically, causing disease: [52] [53] [54] [55]

    • Entamoeba histolytica is the cause of amoebiasis, or amoebic dysentery.
    • Naegleria fowleri (the "brain-eating amoeba") is a fresh-water-native species that can be fatal to humans if introduced through the nose.
    • Acanthamoeba can cause amoebic keratitis and encephalitis in humans.
    • Balamuthia mandrillaris is the cause of (often fatal) granulomatous amoebic meningoencephalitis.
    • Amoeba have been found to harvest and grow the bacteria implicated in plague.
    • Amoebae can likewise play host to microscopic organisms that are pathogenic to people and help in spreading such microbes. Bacterial pathogens (for example, Legionella) can oppose absorption of food when devoured by amoebae. [56]
    • The presently generally utilized and best-explored amoebae that host other organisms are Acanthamoeba castellanii and Dictyostelium discoideum. [57]
    • Microorganisms that can overcome one-celled critters' guards increase a shelter wherein to multiply, where they are shielded from unfriendly outside conditions by their accidental hosts.

    Recent evidence indicates that several Amoebozoa lineages undergo meiosis.

    Orthologs of genes employed in meiosis of sexual eukaryotes have recently been identified in the Acanthamoeba genome. These genes included Spo11, Mre11, Rad50, Rad51, Rad52, Mnd1, Dmc1, Msh and Mlh. [58] This finding suggests that the ‘'Acanthamoeba'’ are capable of some form of meiosis and may be able to undergo sexual reproduction.

    The meiosis-specific recombinase, Dmc1, is required for efficient meiotic homologous recombination, and Dmc1 is expressed in Entamoeba histolytica. [59] The purified Dmc1 from E. histolytica forms presynaptic filaments and catalyses ATP-dependent homologous DNA pairing and DNA strand exchange over at least several thousand base pairs. [59] The DNA pairing and strand exchange reactions are enhanced by the eukaryotic meiosis-specific recombination accessory factor (heterodimer) Hop2-Mnd1. [59] These processes are central to meiotic recombination, suggesting that E. histolytica undergoes meiosis. [59]

    Studies of Entamoeba invadens found that, during the conversion from the tetraploid uninucleate trophozoite to the tetranucleate cyst, homologous recombination is enhanced. [60] Expression of genes with functions related to the major steps of meiotic recombination also increase during encystations. [60] These findings in E. invadens, combined with evidence from studies of E. histolytica indicate the presence of meiosis in the Entamoeba.

    Dictyostelium discoideum in the supergroup Amoebozoa can undergo mating and sexual reproduction including meiosis when food is scarce. [61] [62]

    Since the Amoebozoa diverged early from the eukaryotic family tree, these results suggest that meiosis was present early in eukaryotic evolution. Furthermore, these findings are consistent with the proposal of Lahr et al. [63] that the majority of amoeboid lineages are anciently sexual.


    The yet unknown

    There are caution signals, too. For example, some of the earliest microbial products are selected because they are easy to produce in large scale fermenters and production facilities – not necessarily because benefits are proven in all applications. And there is a challenge in repeatability when every animal and every acre of soil is different. It’s hard to apply standards across live bacterial products and even harder to make them work within a multitude of variability. We’re still learning about complex interactions sometimes, introducing opportunistic microbes might yield unintended consequences.

    Another caution comes from the sheer volume of noise in the market, described as the “Wild West of microbiome science,” in one review in Nature. Many of the new products offer carefully phrased descriptions skirting around benefits proven and implied, such as in the profusion of over-the-counter health and skincare probiotic products.

    With cautions noted, however, I posit this “Wild West” is still worth exploring. Where science is untangling complexity and providing clear pathways to specific outcomes, there’s an opportunity for improved animal health, starting with antibiotic avoidance and better nutrient uptake. The same is true for soil health and developing stronger, more resilient crops. The first Green Revolution relied on chemistry it’s not hard to see that we are entering a new era of better living through biology.


    Watch the video: Plaster Bagworm (January 2022).