Bionomics and Nutritional Ecology of Three Species of Psylloidea of Varied Guilds Feeding on Different Species of Eucalyptus in Central-Western New South Wales

The Australian Psylloidea, comprising about 400 described species, are dominated by a major radiation of the Spondyliaspidinae (Aphalaridae) that has evolved multiple-feeding strategies on the predominantly endemic Australian taxon Eucalyptus (Myrtaceae). In this thesis, I will be presenting the population
dynamics, oviposition behaviour, feeding behaviour and nutritional requirements
of three species of Aphalaridae representing three different feeding guilds: ‘freeliving’ Ctenarytaina eucalypti that infests leaves and tender shoots of Eucalyptus globulus, ‘lerp-forming’ Glycaspis sp. on the mature leaves of Eucalyptus sideroxylon and ‘gall-inducing’ Glycaspis (Synglycaspis) sp. on the developing leaves of E. macrorhyncha in the Orange district of Central-western Tablelands bioregion of south-eastern New South Wales. Chapter 1 pertains to the general introduction of Psylloidea specifically referring to the free-living, lerp-forming and gall-inducing taxa of Australia. Introduction also includes life history pattern of Psylloidea in general and biochemistry of insect and plant interaction.

Bionomics of three selected Aphalaridae
Chapter 2 pertains to bionomics of C. eucalypti on juvenile leaves of E. globulus.
It completes 4—5 generations per year in Orange region and populations in field
conditions occur throughout the year, although numbers were lower in the peak
summer and winter of 2011, 2012 and 2013. The growth ratios were nconsistent and growth ratio for body length of the nymphal instars varied between 1.1 and 1.7. The responses in the juvenile E. globulus leaf tissues to oviposition include subcellular damage with the accumulation of callosic material indicating the earliest response of stress signals in E. globulus to wounding. The feeding action of the first- and second-nymphal instars on the mesophyll cells inflicts both mechanical and chemical damage and manifests poorly differentiated primordialpalisade cells, and autolyzed protoplast. The phloem-feeding third-, fourth-, fifth- nymphal instars and adults induce changes in phloem parenchyma, which occur as actively dividing cells and including autophagic vacuoles. The arrangement of antennal sensilla of C. eucalypti varies among nymphs and adults. The adults and nymphs of C. eucalypti appear to prefer specific leaf parts suggesting that the levels and quality of nutrients vary in the same leaf.

Chapter 3 includes the bionomics of the lerp-forming species of Glycaspis
on the mature leaves of E. sideroxylon. It completes 3—4 generations per year in
Orange region and population trends assessed in 2011―2012 and 2012―2013
showed distinct variations, which have been related to environmental factors.
Developmental events in this taxon do not follow Dyar’s rule strictly and growth
ratio for body length varies between 1.23 and 1.3. Sensillar designs vary between
the nymphal instars and adults. The trichoid sensilla and sensillar cavities on the
antennae perform mechanosensory and olfactory functions. Oviposition induces a non-lethal hypersensitive response in the host leaf. In the 2012—2013 sampling, oviposition occurred preferentially proximally to leaf cracks which could be a strategy of this taxon to establish an ‘easier’ access to moisture. This taxon constructs lerps with anal sugary exudates first building the ribs of the lerps and later filling the space between the polymerized ribs with horizontal tractions of the same sugary substance.

Chapter 4 pertains to the bionomics of species of Glycaspis (Synglycaspis) that induces spherical, ostiolate galls on the leaves of E. macrorhyncha. This species completes two generations in a year and the immature stages show inconsistent growth rates, not conforming to Dyar’s rule, with the growth rate of body length varying from 1.3 to 2. The rhinaria near the apices of flagellomeres in the adult antennae of Glycaspis (Synglycaspis) sp. are highly likely to be used as chemoreceptors directly in host selection. Oviposition elicits a passive response in the leaves of E. macrorhyncha. The host tissue not only supplies water, but also nutrients to the developing embryo via the embedded pedicel. Feeding action of the first nymphal instar initiates the gall and morphogenetic gradients. The gall attains its near final spherical shape during the second nymphal instar and this spherical gall remains plugged with sugary material, which all the immature stages secrete from the last abdominal segment. The first and second nymphal instars feed on the newly differentiated parenchyma tissue in galls, whereas the third, fourth, and fifth instars feed on phloem. Consequently the multi-layer parenchymatous nutritive tissue that initially develops lining the nymphal chamber dries and shrivels, which induces a modest expansive growth of the gall.

Nutritional physiology of three selected Aphalaridae
To characterise the role of salivary proteins of Psylloidea in sylloidea―Eucalyptus interaction, proteins in salivary glands of C. eucalypti and phloem exudate of E. globulus were analysed. Due to time constraints, the labour involving task of dissecting salivary gland of Glycaspis sp. and Glycaspis (Synglycaspis) sp. could not be done. Chapter 5 pertains to the analysis of the structure of salivary glands of C. eucalypti. The principal salivary gland is multilobed, whereas the accessory gland is tubular. 1—D electrophoresis revealed proteins of approximately 58 and 64 kDa in the salivary gland extracts and proteins of similar molecular weights in the extracted phloem exudates from
infested leaves and tender shoots of E. globulus. Proteins that possibly fall within this range include, but not limited to, are glucose–methanol–choline–
oxidoreductase (53―66 kDa), Zn–binding dehydrogenase (67 kDa), and esterase
(65―96 kDa), in addition to cytochrome P–450 (50―55 kDa), trehalase (56
kDa), amylase (50―75 kDa), and lipase (48―52 kDa). Previous studies indicate
that the glucose–methanol–choline–oxidoreductase, Zn–binding dehydrogenase,
cytochrome P-450 and trehalase suppress plant-defense mechanism, whereas the cell-degrading enzymes such as amylase, lipase, esterase have a possible role in enabling C. eucalypti’s stylet insertion into leaf and shoot tissues of E. globulus.

Chapter 6 includes the analysis of the variation in the nutritional
requirements of the Aphalaridae belonging to distinctly different feeding guilds
on three species of Eucalyptus. Levels of total non structural carbohydrates, δ13C, δ15N, amino acids and fatty acids were analysed. TNC levels and the ratios between δ13C and δ15N were maximum in the leaves of E. macrorhyncha infested by the gall-inducing species Glycaspis (Synglycaspis) sp., followed by that in the leaves of E. sideroxylon infested by the lerp-forming Glycaspis sp., and the lowest occurred in the leaves of E. globulus infested by the free-living C.
eucalypti. Whereas levels of δ15N, total mass of amino acids, total mass of fatty
acids, and total mineral percentage were maximum in the C. eucalypti—E.
globulus interacting system, followed by Glycaspis sp.—E. sideroxylon system,
and the minimum in the Glycaspis (Synglycaspis) sp.—E. macrorhyncha system.
The free-living C. eucalypti appear to stress the leaves of E. globulus more intensely by its group-feeding behavior, whereas the gall-inducing Glycaspis
(Synglycaspis) sp. essentially induces the host to respond with a novel phenotypic expression, viz., the gall. The lerp-forming Glycaspis (Synglycaspis) sp. imbibes nutrients, especially sugars of which the excess is secreted in the form of its characteristic lerp.

Plants include a variety of lipids which enable storing of energy in cell
membranes. In insects, cholesterol reinforces cell-membrane integrity and
synthesizes moulting hormones but insects, in general, cannot synthesize
cholesterol without a steroid precursor from their hosts. Plant feeding insects
utilize different kinds of phytosterols to synthesize ecdysteroids. Chapter 7
pertains to the analysis of complex lipids and steroid precursors in three
interacting system of Aphalaridae and Eucalyptus. We also selected Eucalyptus
rossii and Eucalyptus dives as the ‘closest’ relatives of E. macrorhyncha in the
Orange subregion to compare complex lipids and steroid precursors in young and mature leaves of these two species with those in the young and mature leaves of E. macrorhyncha. The pattern of changes in complex lipids and sterols in uninfested and infested leaves of E. macrorhyncha hosting Glycaspis
(Synglycaspis) sp., E. sideroxylon hosting Glycaspis sp. and E. globulus hosting
C. eucalypti show that different lipid occurrences in plant tissues is influenced by
group and solitary feeding. The low levels of MGDG (mono-galactosyldiacylglycerides) and DGDG (di- galactosyldi-acylglycerides) levels in E. macrorhyncha could be a key factor in regulating the preference and selection of E. macrorhyncha by Glycaspis (Synglycaspis). The levels of ergosterol increase
in galls that include the second, third and fourth nymphal instars and decrease in galls that include the fifth nymphal instars of Glycaspis (Synglycaspis) sp.,
indicating that this sterol is produced in host tissues during gall development and is minimised when the insect is preparing to moult into adult and exit the gall. The high levels of the sterol 440.3 in the young leaves of E. macrorhyncha
indicate the possibility of this sterol being a critical factor in the selection of
young leaves of E. macrorhyncha by the gravid female of Glycaspis
(Synglycaspis) sp. to commence oviposition and subsequent gall development.

General discussion and conclusion
Chapter 8 includes a general discussion of the bionomics and nutritional
physiology of selected guilds of Australian Aphalaridae which demonstrate that
the two glycaspids, species of Glycaspis and species of Glycaspis (Synglycaspis)
show similar patterns in their population dynamics and oviposition behaviour.
The species of Glycaspis and species of Glycaspis (Synglycaspis) consume
greater levels of nutrients, especially sugars, compared with that of C. eucalypti.
This behaviour appears reasonable given that these species have a commitment to generate either a lerp made of sugars or a gall generated by the plant induced by the stimulus from Glycaspis (Synglycapis). The free-living C. eucalypti
nonetheless appear to be better adapted to different climatic regions and groupfeeding behaviour. The levels of ergosterol and sterol 440.3 indicate them that they could be responsible for making E. macrorhyncha susceptible for gallinducing Glycaspis (Synglycaspis) sp.

The variation in the bionomics of three selected species of Aphalaridae appears to be influenced by their nutritional requirements. The feeding behaviour, such as solitary and group feeding, also plays a major role in their nutritional biology.