Table Of Content"Antifungal defenses in subterranean termites
and Cryptocercus woodroaches"
By: Diandra Denier
A thesis in partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE
Towson Biology Department
Towson University
8000 York Rd
Towson, MD 21252
January 6, 2013
ii
Acknowledgments
Thanks to Advisor Mark Bulmer, Ph.D for guidance and support throughout the thesis
process, committee members Michelle Snyder, Ph.D and Roland Roberts, Ph.D for
involvement in editing the thesis proposal and defense, Towson Graduate and fellow lab
member Casey Hamilton for help with development of methods used in this thesis, Lab
members Frank Lay and Joe Velenovsky for contributions to termite and woodroach
collection as well as work with GNBPs and Roxy Cavey for testing preliminary methods
for identifying and cultivating Metarhizium.
Special thanks to my supportive boyfriend, Kenneth Kirwan, for always being willing to
lend an ear and listen to my research. Also thanks to my family for being behind me
every step of the way.
iii
Abstract
Diandra Denier
The secreted β-1,3-glucanase activity of Gram-negative bacteria binding proteins
(GNBPs) provides woodroaches with important prophylactic protection from fungal
pathogens such as Metarhizium anisopliae. Cuticular washes have antifungal activity
against M. anisopliae conidia that was suppressed by an inhibitor (GDL) of termite
GNBP β-1,3- glucanase activity. Cryptocercus punctulatus nymphs that were treated with
GDL and subsequently exposed to M. anisopliae conidia show significantly greater
mortality than the untreated nymphs exposed to conidia. The β-1,3- glucanase activity of
GNBPs therefore appears to be critical for protecting Cryptocercus woodroaches from
fungal pathogens.
Analysis of local and foreign Metarhizium strains indicates that Metarhizium has
the potential to influence the evolution of the termite immune system. To investigate
Metarhizium strain variety and virulence, six strains were isolated and identified from
nearby Reticulitermes flavipes collection sites. Colonies varied significantly in their
susceptibility to the six isolates of Metarhizium, which were collected across a rough
transect of approximately 1 km. These fungal isolates represented three separate species,
M. brunneum, M. robertsii and M. guizhouense. There was a significant correlation
between the genetic distance between isolates and their difference in virulence in three of
iv
four termite colonies. This variety of Metarhizium over small spatial scales suggests that
adaptive evolution in the termite immune system may arise as a result of a virulent
Metarhizium strain periodically creating epizootics that are countered by the evolution of
resistance in the host.
Messenger RNA sequences of Gram Negative Bacteria-binding Protein 1
(GNBP1) were identified and analyzed in two species of subterranean termites. Using
population genetic methods, comparisons were made between this gene and two
additional antifungal genes in the subterranean termites and two species of woodroaches,
Cryptocercus punctulatus and Cryptocercus wrighti. An analysis of nucleotide
intraspecific polymorphism indicated that these genes frequently face selective sweeps,
possibly as a result of a virulent fungal strain spreading through populations and selecting
for resistant specific alleles that afford the greatest resistance to infection.
v
Table of Contents
Chapter 1. A common antifungal defense strategy in Pg 1
Cryptocercus woodroaches & termites
Abstract ….............................................................................. 1
Introduction ............................................................................ 2
Materials & Methods .............................................................. 4
Woodroach collection
Metarhizium anisopliae isolation
β-1,3-Glucanase activity of cuticular washes 5
In vitro antifungal assay of cuticular washes
In vivo antifungal assay
Results …................................................................................. 7
β-1,3-Glucanase & antifungal activity
GDL β-1,3-glucanase inhibition & 9
nymph survival
Discussion …............................................................................ 11
Chapter 2. Variation in subterranean termite Pg 13
susceptibility to indigenous Metarhizium species
Abstract ….............................................................................. 13
Introduction …........................................................................ 14
Materials & Methods ….......................................................... 16
Fungal Isolation
Molecular methodology
In vivo assay 17
Results …............................................................................... 20
Phylogenies & Species Identification
In vivo analysis
Discussion …......................................................................... 25
vi
Chapter 3. A comparison of the selective pressure on Pg 27
antifungal genes in Cryptocercus woodroaches & termites
Abstract …............................................................................. 27
Introduction …....................................................................... 28
Materials & Methods …........................................................ 31
Insect & fungus sample preparation
Isolation & characterization of GNBP1 32
Statistical analysis
Results ….............................................................................. 33
Discussion …........................................................................ 36
References …............................................................................................ 38
CV …............................................................................................................ 43
vii
List of Tables
Chapter 2
Table 1. BLAST identities of EF1 sequence Pg 21
Table 2. Correlations between differences in genetic ….............................. 24
distance and hazard ratios of survivorship for
different Metarhizium isolates in four colonies of R. flavipes.
Chapter 3
Table 1. Measures of Tajima’s D for GNBP1, GNBP2 Pg 34
and termicin.
Table 2. McDonald-Kreitman test of GNBP1, GNBP2 …........................... 35
and termicin in Cryptocercus.
Table 3. McDonald-Kreitman test of GNBP1, GNBP2 …........................... 35
and termicin in Reticulitermes
viii
List of Figures
Chapter 1
Figure 1. CM-curdlan-RBB polyacrylamide gels. Pg 8
a) C. punctulatus cuticular washes, b) C. punctulatus
tissue extracts.
Figure 2. Colony-forming units of M. anisopliae …..................................... 8
conidia after treatment with C. punctulatis cuticular
0.1 % Tween 80 washes (Cp), 0.1 % Tween 80 alone
(control) or washes and 100 mM GDL (Cp GDL)
Figure 3. Woodroach survival after GDL and …........................................ 10
M. anisopliae exposure
Chapter 2
Figure 1. Map showing the genetic distances Pg 18
between the different Metarhizium isolates used in the study.
Figure 2. Maximum-likelihood tree with bootstrap …...........................…. 20
values for the concatenated IGS and 16S sequence.
Figure 3. Maximum-likelihood tree with bootstrap …...........................…. 21
values for the 5’ region of EF1.
Figure 4. Cox regression analysis of survivorship …................................ 22
worker termites from four termite colonies challenged
with six fungal isolates of Metarhizium over the course of 21 days.
ix
Antifungal Defense 1
Chapter 1
A common antifungal defense strategy in Cryptocercus
woodroaches and termites
Abstract
In termites, the secreted β-1,3-glucanase activity of Gram-negative bacteria binding
proteins (GNBPs) provides important prophylactic protection from fungal pathogens such
as Metarhizium anisopliae, which can evade the immune system after entering the insect.
Termites evolved from a cockroach-like ancestor that is believed to resemble
Cryptocercus woodroaches. Here, β-1,3-glucanase activity is identified on the cuticular
surface of the woodroach Cryptocercus punctulatus that originates from the salivary
gland and is likely spread by allogrooming. Cuticular washes have antifungal activity
against M. anisopliae conidia that is suppressed by an inhibitor (GDL) of termite GNBP
β-1,3- glucanase activity. C. punctulatus nymphs that are treated with GDL and
subsequently exposed to M. anisopliae conidia show significantly greater mortality than
the untreated nymphs exposed to conidia.
Description:Metarhizium strain periodically creating epizootics that are countered by the evolution of resistance in the additional antifungal genes in the subterranean termites and two species of woodroaches, .. reproduce then the pathogen may often have the upper hand in this race as it typically has a shor
