**2. Insect eggshell morphology**

Insect eggs are remarkably structured to provide the developing insect embryo with protec‐ tion, simultaneously providing a barrier against insecticide penetration. This is evident from the array of structural designs observed on eggshells across insect species, which have specifically evolved to provide the developing insect ultimate protection in the environment. There are many variations in the respiratory structures, shape, size, coloration, and the chorionic structure of eggs, all seemingly evolved to withstand environmental stress. The diversity of eggshells and the structural complexity of the eggshell (Figure 1) are evident in three butterfly eggs in the families Nymphalidae and Saturniidae. The amazing diversity of insect eggs has received little attention from the scientific community. H. E. Hinton's threevolume work on the biology of insect eggs [1] represents one of the most significant contribu‐ tions to understanding insect eggs.

Various studies have attempted to characterize the insect eggshell, or chorion, and its layers. Eggshell morphological descriptions have revealed that many insect eggs have detailed sculpturing on the outside of the eggshell. The sculpturing is usually comprised of multisided geometrical shapes, arranged on the eggshell in an aesthetically pleasing pattern.

Not all insect eggshells are perfectly symmetrical. For instance, the outer surfaces of the chorion in true bugs oftentimes are geometrical but the shapes are irregular in shape and size. Furthermore, some insect eggs lack the geometrical shapes extending from the eggshell and are completely smooth. The formation and shape of the eggshell is highly dependent on the outline shape of the mother's follicle cells that synthesize the eggshell.

In the family Pentatomidae (stink bugs), the chorion is characterized by the surface structure, termed either "spinose" or "coarse" [2]. "Spinose" refers to insect eggs that have projections arranged in patterns that extend outwardly from the surface. The term "coarse" refers to indented pit structures on the outer eggshell surface [2].

The chorion is produced within the female's ovariole by the follicular epithelium. In the simplest form, the chorion is typically comprised of three layers (exochorion, endochorion

Insect eggs have adapted mechanisms to enhance their survival, including the enclosure of the embryo within an eggshell. The eggshell, also referred to as the chorion, is the first line of defense for the developing embryo against environmental stressors. One main environmental stressor that pest insect species have to face is human use of pesticides. The unique structure

Insecticides that do penetrate the eggshell have to reach their target site within the embryo to be effective. A few insects have been shown to have developed enzymatic resistance to insecticides in the egg stage. Eggs treated with insecticides repeatedly were shown to produce high levels of enzymatic activity to break down insecticides. The combination of reduced penetration through the eggshell and pesticide resistance makes eggs an extremely difficult

Insect eggs are remarkably structured to provide the developing insect embryo with protec‐ tion, simultaneously providing a barrier against insecticide penetration. This is evident from the array of structural designs observed on eggshells across insect species, which have specifically evolved to provide the developing insect ultimate protection in the environment. There are many variations in the respiratory structures, shape, size, coloration, and the chorionic structure of eggs, all seemingly evolved to withstand environmental stress. The diversity of eggshells and the structural complexity of the eggshell (Figure 1) are evident in three butterfly eggs in the families Nymphalidae and Saturniidae. The amazing diversity of insect eggs has received little attention from the scientific community. H. E. Hinton's threevolume work on the biology of insect eggs [1] represents one of the most significant contribu‐

Various studies have attempted to characterize the insect eggshell, or chorion, and its layers. Eggshell morphological descriptions have revealed that many insect eggs have detailed sculpturing on the outside of the eggshell. The sculpturing is usually comprised of multisided

Not all insect eggshells are perfectly symmetrical. For instance, the outer surfaces of the chorion in true bugs oftentimes are geometrical but the shapes are irregular in shape and size. Furthermore, some insect eggs lack the geometrical shapes extending from the eggshell and are completely smooth. The formation and shape of the eggshell is highly dependent on the

In the family Pentatomidae (stink bugs), the chorion is characterized by the surface structure, termed either "spinose" or "coarse" [2]. "Spinose" refers to insect eggs that have projections arranged in patterns that extend outwardly from the surface. The term "coarse" refers to

The chorion is produced within the female's ovariole by the follicular epithelium. In the simplest form, the chorion is typically comprised of three layers (exochorion, endochorion

geometrical shapes, arranged on the eggshell in an aesthetically pleasing pattern.

outline shape of the mother's follicle cells that synthesize the eggshell.

indented pit structures on the outer eggshell surface [2].

of the eggshell renders most insecticidal products impenetrable through the eggshell.

life stage to kill.

84 Insecticides Resistance

**2. Insect eggshell morphology**

tions to understanding insect eggs.

**Figure 1.** Automontage photographs of three butterfly eggs. (A) An egg from the gulf fritillary, *Agraulis vanillae*; fami‐ ly: Nymphalidae. (B) Eggs from the luna moth, *Actias luna*; family: Saturniidae. Note the exuvia from the recent molt of the larvae. (C) Eggs from the zebra longwing, *Heliconius charithonia*; family: Nymphalidae.

[inner and outer], and vitelline membrane) [2, 3]. The vitelline membrane is the innermost layer that surrounds the embryo. A few insect studies have further subdivided the eggshell layers into waxy layers and crystal chorionic layers, which most probably serve as the main barriers in the eggshell against water loss. However, these structures and layers differ between insect families and species, depending on their habitat and individual respiratory and water requirements.

Gravid female insects have accessory glands that secrete gluelike substances, sometimes referred to as "cement" to adhere their oviposited eggs to a substrate. These glue substances have to be able to withstand environmental stress for the duration of embryonic development before the larvae is ready to emerge from the eggshell. Much like the eggshell, these glue substances are primarily comprised of proteins [4]. When considering the penetration of insecticides, the glue sheath adds an extra layer of protection for the developing embryo. The head louse glue sheath is especially problematic from the perspective of lice control.

Anyone who has experienced head lice is well aware of the glue sheath that surrounds a head louse egg, called a nit. The glue sheath laid down by the mother from her collateral glands adheres the nit to the hair shaft. This makes nits incredibly difficult to remove from hair. The nit comb, which is a widely used management technique for head lice, has very fine teeth designed to brush nits from the hair shaft. The sheath covers the entire egg, except at the operculum where respiration occurs [5]. Understanding the components of the sheath could allow researchers to develop novel ways to denature the protein components of the sheath or to coat the sheath and prevent the embryo from breathing [5].

The embryo inside of the eggshell hatches, or encloses, through the egg cap, called the operculum. The operculum is usually located on the anterior pole of the egg. The border of the operculum is comprised of multiple, small, uniform-shaped holes along the circumference of an egg, usually aligned side by side. The appearance of these holes is similar to a loose-leaf notebook with a perforated edge on each page that allows pages to easily be torn out. The perforations on the egg make the operculum easier to break open, thus allowing the first instar larvae to push through the operculum during eclosion. This process is taxing to the small larvae, so many larvae have a specialized spine, or egg burster, on their heads to assist with hatching from the operculum [1]. In addition to an egg burster, the larvae will grow in size by engulfing air and amniotic fluid, creating pressure inside of the egg until they expand enough to break free from the eggshell.

In addition to the eggshell layers, there are structures present on the eggshell for respiration (aeropyles) and fertilization (micropyles) and also inner eggshell structures for the movement of oxygen (pillars, sometimes also referred to as struts or columns). The pillar, or column structures, can be observed easily in the scanning electron micrograph of a hatched bed bug egg (Figure 2). These structures that open into the eggshell are potentially sites that would allow insecticides to enter the insect egg. The insect eggshell must maximize embryo respira‐ tion while preventing water loss. The eggshell is designed not only to limit water loss from the egg but also to limit excessive water from entering the eggshell. Consequently, water-based insecticidal preparations do not easily penetrate insect eggshells.

**Figure 2.** Scanning electron micrograph of a hatched bed bug egg. The egg has been cut in half, and the operculum has been removed for visualization purposes. The inner layers, including the respiratory struts and columns, can be ob‐ served.

Insect eggs are very small; therefore, they have an increased surface area-to-volume ratio [6]. Having a large proportion of the insect egg's surface exposed to the environment in relation to the internal volume makes the egg even more prone to desiccation. Multiple studies have been conducted to quantify the balance between respiration and water conservation of insect eggs [6–8]
