George Happ - Insect Research 

Our research over the period from 1959 through 1995 was in the field of insect physiology.

Insect Research (see Publications)

Our research on insects started from chemical ecology and behavior and ultimately focused on  molecular physiology.

The insect trail began with allomones and pheromones.

Allomones:

Using cytochemistry and electron microscopy, we asked how insect defensive glands could make highly reactive cellular toxicants without self-poisoning.  In several species with epidermal defensive glands, the special permeability properties of the cuticle are the key to self-protection.  As demonstrated by Tom Eisner and others, fine cuticular plumbing runs from minuscule reaction vesicles bounded by the microvilli of the individual secretory cells.  We traced non-toxic precursors into the reaction compartments where they are transformed into poisonous toxicants.   The reaction products are conveyed through cuticular ductules to impermeable cuticular storage reservoirs.
Next, we used insect defensive glands to explore the metabolic scope of the insect.  In mammals, terpenes and steroids are made from isoprene units derived from mevalonic acid.  It was then known that insects could not make steroids, and yet they used terpenes as defensive compounds.  In studies of the biosynthesis of  terpenoid defensive allomones, we provided the first strong evidence for the presence of the mevalonic acid pathway in insects.
Allomones also promote symbiosis. As examples of symbiotic associations promoted by insect secretions, we described the ultrastructure of the secretory pockets used by scolytid bark beetles to culture and transport their symbiotic tree-killing fungi.

Pheromones:

By behavioral assays, we revealed the existence of multiple sex pheromones in mealworm beetles  (Tenebrio molitor L.).  A pheromone produced by male beetles attracts females (A) and one produced by females attracts males (B).  When males have been stimulated by female scent, they produce an antiaphrodisiac which inhibits the responses of other males to female scent (C).  Finally, primer pheromones produced by both sexes accelerate reproductive maturation in young adult female (D).
 

Insect Learning:

We showed that insects, trained as larvae by passive avoidance conditioning, went through metamorphosis and showed retention of the conditioning as adult beetles.
 

Mechanisms promoting sperm transfer:

  Sperm transfer in mealworm beetles involves: 1) paired accessory reproductive glands of the male,  2) the spermatophore, a multilayered secretory product of the male glands that packages the sperm for transfer to the female, 3) a female sperm storage organ, the cuticular spermatheca, and 4) the spermathecal accessory gland in the female.   The smaller tubular pair of male accessory reproductive glands are called the TAGs.  The larger pair of male glands, termed the BAGs, contain 8 types of secretory cells that are arranged in a precise pattern. Each patch of secretory cells in the accessory gland  produces cell-specific antigens that are built into particular layers of the spermatophore. The secretory product from the BAG forms a semisolid plug in the lumen  All the cell-specific secretions remain distinct in the semisolid product, as shown for the secretions 7s, 6s, 4s, 3s, and 2s derived from cells 7, 6, 4, 3, and 2 respectively, in the micrograph.  Secretions from the bilateral glands converge in the ejaculatory duct and therein are molded by contractions of a heavy muscular coat into the multilayered wall of the spermatophore. 

  We described the elaborate structure of the spermatophore and its contortions as sperm are expelled. The spermatophore has many layers, some of which are seen in the electron micrograph.  The outside of the spermatophore is at the upper left and the lumen (where sperm are sequestered) is at the lower right.  Once placed within the female reproductive tract, the spermatophore undergoes several programmed extensions as the sperm mass flows forward, a bladder-like anterior expansion swells and within 7 minutes, it explosively ruptures to liberate sperm. 

We isolated, characterized, and sequenced diverse proteins of the male accessory glands. Some of these proteins are unique structural molecules, functionally similar to structural proteins of cuticle or eggshells.  Some are enzymes, including an unusual trehalase which is also a sensitive indicator of terminal differentiation.  Other proteins have highly repetitive amino acid sequences suggesting binding pockets for small molecules. One class of these binding proteins appears to be homologous to pheromone-binding proteins of flies, moths, and hamsters.

Ecdysteroids control reproductive maturation, cell cycling, and differentiation:  

From zero-day pupae (A) to ecdysis 9 days later (B) to 8-day adult (C), male accessory glands grow and differentiate. The maturation of the TAGs and BAGs proceeds along a  precise developmental timetable which is  dependent upon changes in ecdysteroid concentrations. The male glands develop in response to pupal ecdysterone peaks that first control cell cycling in the early pupa and next promote the commitment to terminal differentiation in the late pupa. 

This beetle system has proved to be an excellent model  for the study of sperm transfer and hormonal modulation of reproductive maturation in insects.