PHYLOGENETIC PLACEMENT OF THE WHITE COCONUT SCALE, PARLAGENA BENNETTI WILLIAMS (HEMIPTERA DIASPIDIDAE)

Parlagena bennetti Williams (Hemiptera: Diaspididae) is commonly known as the coconut scale and has only been collected in some islands in the Caribbean, Central America and the northernmost countries of South America. The species P. bennetti has been placed in Parlagena, a genus of few species currently considered as closely related to Parlatoria Targioni Tozzetti, but it has never been involved in molecular phylogenetic analysis. Here we include data from three genes of P. bennetti with 32 other armored scale insects and one outgroup to determine the correct placement of this species among armored scale insects. Both combined analysis and individual genealogies demonstrate the probable placement of this species in the subfamily Diaspidinae, likely as part of the tribe Lepidosaphidini

Investigating endosymbionts of scale insects from the family Diaspididae

Armored scale insects have long been known to harbor endosymbionts, but until recently the endosymbionts have remained unidentified. Using DNA data I have identified the primary endosymbiont as a bacterium from the phylum Bacteriodetes. To accomplish this, I amplified DNA sequences from two genes of the endosymbiont and use these sequences (16S and 23S, 2105 total base pairs), along with previously published sequences from the armored scale hosts (elongation factor 1α and 28S rDNA) to investigate phylogenetic congruence between the two clades. The Bayesian tree for the bacteria is roughly congruent with that of the hosts, with 67% of nodes identical. The high level of congruence between the topologies indicates that these Bacteroidetes are the primary endosymbionts of armored scale insects. To investigate the phylogenetic affinities of these endosymbionts, I aligned some of their 16S rDNA sequences with other known Bacteroidetes endosymbionts and with other similar sequences identified by BLAST searches. I found these endosymbionts to be closely related to bacteria associated with eriococcid and margarodid scale insects, and enodosymbionts of cockroaches and auchenorrynchan insects, and proposed the name Candidatus Uzinura diaspidicola for the primary endosymbionts of armored scale insects. I have also investigated secondary endosymbionts found in the armored scale insect species Aspidiotus nerii. A host manipulating endosymbiont similar to Wolbachia, Cardinium was found in Aspidiotus nerii and there is circumstantial evidence that Cardinium is responsible for inducing parthenogenesis in Aspidiotus nerii. Using PCR and sequencing of 16S rDNA, we have tested 593 individuals and obtained positive PCR results in 67 individuals of 34 populations representing 20. A phylogenetic analysis including all known insect-associated Cardinium 16S sequences shows patterns of horizontal transmission of this endosymbiont among insects. Lastly, I tested molecular evolution rates of Uninura from sexual and parthenogenetic populations of Aspidiotus nerii using five loci: two host nuclear loci, one mtDNA loci, and two symbiont loci. I hypothesized that the symbionts and mitochondria would evolve at similar rates, however, though the endosymbionts are vertically transmitted and experience similar bottlenecks to the mitochondria, the data suggest that they evolve at similar or slower rates to the nuclear DNA

Distribution of the Primary Endosymbiont (Candidatus Uzinura Diaspidicola) Within Host Insects from the Scale Insect Family Diaspididae

It has long been known that armored scale insects harbor endosymbiotic bacteria inside specialized cells called bacteriocytes. Originally, these endosymbionts were thought to be fungal symbionts but they are now known to be bacterial and have been named Uzinura diaspidicola. Bacteriocyte and endosymbiont distribution patterns within host insects were visualized using in situ hybridization via 16S rRNA specific probes. Images of scale insect embryos, eggs and adult scale insects show patterns of localized bacteriocytes in embryos and randomly distributed bacteriocytes in adults. The symbiont pocket was not found in the armored scale insect eggs that were tested. The pattern of dispersed bacteriocytes in adult scale insects suggest that Uzinura and Blattabacteria may share some homologous traits that coincide with similar life style requirements, such as dispersal in fat bodies and uric acid recycling

Evolutionary Relationships among Primary Endosymbionts of the Mealybug Subfamily Phenacoccinae (Hemiptera: Coccoidea: Pseudococcidae) ▿

Mealybugs (Coccoidea: Pseudococcidae) are sap-sucking plant parasites that harbor bacterial endosymbionts within specialized organs. Previous studies have identified two subfamilies, Pseudococcinae and Phenacoccinae, within mealybugs and determined the primary endosymbionts (P-endosymbionts) of the Pseudococcinae to be Betaproteobacteria (“Candidatus Tremblaya princeps”) containing Gammaproteobacteria secondary symbionts. Here, the P-endosymbionts of phenacoccine mealybugs are characterized based on 16S rRNA from the bacteria of 20 species of phenacoccine mealybugs and four outgroup Puto species (Coccoidea: Putoidae) and aligned to more than 100 published 16S rRNA sequences from symbiotic and free-living bacteria. Phylogenetic analyses recovered three separate lineages of bacteria from the Phenacoccinae, and these are considered to be the P-endosymbionts of their respective mealybug hosts, with those from (i) the mealybug genus Rastrococcus belonging to the Bacteroidetes, (ii) the subterranean mealybugs, tribe Rhizoecini, also within Bacteroidetes, in a clade sister to cockroach endosymbionts (Blattabacterium), and (iii) the remaining Phenacoccinae within the Betaproteobacteria, forming a well-supported sister group to “Candidatus Tremblaya princeps.” Names are proposed for two strongly supported lineages: “Candidatus Brownia rhizoecola” for P-endosymbionts of Rhizoecini and “Candidatus Tremblaya phenacola” for P-endosymbionts of Phenacoccinae excluding Rastrococcus and Rhizoecini. Rates of nucleotide substitution among lineages of Tremblaya were inferred to be significantly faster than those of free-living Betaproteobacteria. Analyses also recovered a clade of Gammaproteobacteria, sister to the P-endosymbiont lineage of aphids (“Candidatus Buchnera aphidicola”), containing the endosymbionts of Putoidae, the secondary endosymbionts of pseudococcine mealybugs, and the endosymbionts of several other insect groups

Sex ratio at emergence.

<p>Block diagram of percent male to female <i>T. frequens</i>, scaled to 100%, for three sampling months. All months show an excess of females (dark grey), and proportions are very similar throughout.</p

Fluctuations of adult females and tenerals throughout 24 hrs.

<p>Block diagram of the average numbers of depositing females, and tenerals of <i>T. frequens</i> from glue board time series on the pupal field, with standard error bars (95% confidence interval). Timeframe 1 (1715 hrs until 2315 hrs) shows high activity of depositing females, timeframe 2 (2315 hrs until 1015 hrs) represents the peak of teneral emergence, while almost no activity is observed during timeframe 3 (1015 hrs until 1715 hrs) for both tenerals, and depositing females.</p

Methods of data capture.

<p>(<b>A</b>) Excerpt of temperature loggers (LogTag, Haxo 8), for 4 measuring points throughout the cave. T1 represents measurements in the main roost (room III); T4, and 6, are placed in the entrance area of room I; T5 represents the measurements on the pupal field (as per cave outline on the right). Graph was modified from Dittmar and Mayberry <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019438#pone.0019438-Dittmar3" target="_blank">[46]</a>. Standard deviations are shown for each logger. (<b>B</b>) Overview of a glue board, placed on the pupal field. Dark spots are captured bat flies.</p

Schematic diagram comparing parasite demography on-host (right panel), and off-host (left panel).

<p><b><u>On-host</u></b> a physical ratio of 1∶1 is observed. Red rectangle represents females with developing offspring, which are not available for reproduction. Females within green rectangle are a mixture of returning females, and teneral females (N). The green rectangles (male and female) on-host represent the operational sex ratio (OSR), which is likely male biased. <b><u>Off-host</u></b>, depositing females leave the hosts (1), and arrive at the pupal field (2), where they give birth to their offspring. Some die due to predation (skull), while new females and males emerge leave the field (3) and return to the general host population (4). Mating ensues, and females become unavailable for mating. The ratio of males and females at emergence is skewed towards females.</p

Schematic overview of bat fly location during 24 hrs.

<p>Location of adult bat flies, teneral flies, and bat hosts during the three sampling timeframes (1,2,3) throughout a day is shown (A,B,C). (<b>A</b>) During Timeframe III almost no host activity can be detected, and an equal sex ratio is observed on roosting hosts, representing the physical true population ratio. (<b>B</b>) During Timeframe II bat hosts are exiting the roost, and male skewed parasite ratios can be observed on exiting foraging bats (subpopulation). Female depositing flies can be observed on the pupal field. No males are observed in this area. Some males and females are observed in the roost, without bat hosts. (<b>C</b>) Timeframe I, is marked by the emergence of tenerals from the pupae, which show a female biased sex differential. The cave outline was extracted from the current Culebrones cave map (Sociedad Espeleologica de Puerto Rico, Suunto and nylon tape survey, 2007), and only shows the first two rooms of the cave. The back area (III) is roost to five other species of bats known from this cave.</p

Predation on pupal field.

<p>(<b>A</b>) Pupa on the deposition field attacked by a fungus. (<b>B</b>) Debris of bat fly wings (from predation), and fungi in front of a nest entrance of <i>Tetramorium</i> sp. (<b>C</b>) <i>Tetramorium</i> worker ant capturing a freshly emerged teneral <i>Trichobius frequens</i> (see unfurled wing tips). (<b>D</b>) <i>Tetramorium</i> worker ant capturing a female <i>T. frequens</i> after larviposition.</p