**3. Respiration and water conservation of insect eggs**

allow researchers to develop novel ways to denature the protein components of the sheath or

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

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

**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‐

to coat the sheath and prevent the embryo from breathing [5].

insecticidal preparations do not easily penetrate insect eggshells.

to break free from the eggshell.

86 Insecticides Resistance

served.

The respiratory systems in insect eggs are very different from systems in other insect life stages. The principle idea is still the same; there must be openings on the eggshell to allow the exchange of gases with the atmosphere and mechanisms to move gases throughout the eggshell. Typically, insect embryos respire through tiny pores called aeropyles located on the outer eggshell, which allows for efficient gas exchange and reduces water loss during respi‐ ratory activities (Figure 3). To reduce water loss, many terrestrial insect eggs have a reduced numbers of aeropyles [6]. The aeropyles connect to an inner air-filled space that is referred to as the pillar system, which moves oxygen from the outer eggshell layers to the inner embryo. Insect eggs may have their aeropyles located on the outer rim of the operculum, as seen on many hemipteran eggs, or, if there is no defined rim, they will be located on the border of the operculum. Alternatively, aeropyles can also be scattered across the egg surface.

**Figure 3.** Respiratory system of the common house fly. Aeropyles can be seen extending through the eggshell and the various columnar sections and meshwork that compose the entire respiratory structure (Hinton 1967 [7]).

Considering there are an estimated 1 million insects on earth, there are numerous modifica‐ tions and exceptions to the respiratory systems of insect eggs dependent upon the environment and respiratory requirements of individual species. While terrestrial insects have an inner pillar system, aquatic insect eggs primarily respire with a plastron. The plastron is a gas filled air layer below the outer chorion of the egg shell. The plastron acts as a physical gill that allows eggs to respire under water [6]

Some terrestrial insect eggs that are oviposited in environments flooded with water may also have a plastron. In eggs with a large plastron membrane, only small parts of the plastron are permeable, limiting water loss. Terrestrial eggs typically do not have a plastron but do have a gas layer directly under the outer surface of the chorion and connected to the aeropyles. Aeropyles of terrestrial insects occasionally flooded by rainwater are located on the ends of respiratory horns. These horns extend above the water surface to allow for more efficient uptake of oxygen when the egg is surrounded by a layer of water [6]. These respiratory horns are essential to allow the insect to breathe during periods of heavy rain when an insect egg lacks the plastron respiratory system.

The eggshell layers and elaborate respiratory systems of insect eggs are modified to conserve water for the insect egg. Terrestrial insect eggs are provided from their mother all of the water necessary for survival and development at the time of oviposition. Therefore, the embryos must conserve this limited amount of water during their development while also respiring. Water loss occurs across the chorion and is correlated to oxygen consumption requirements of the embryo. The more gas exchange that occurs, the more vulnerable the insect embryo is to water loss.

The waxy layer in the insect eggshell most probably is the main barrier in the eggshell that prevents water loss. There was a significant increase in water loss in *Manduca sexta* eggs when the waxy layer was dissolved by organic solvents [8]. In addition to the waxy layer, the serosal membrane that envelopes the embryo has been shown to also protect insect eggs against desiccation [9]. RNAi technology has been used to prevent the development of the serosa in the beetle *Tribolium castaneum* [9]. Following RNAi treatment, the serosa-less eggs were subjected to a range of different humidities. Eggs exposed to the lowest humidities could not retain water and fewer eggs hatched compared to eggs that still had their serosal membrane intact.

Environmental factors and embryonic development can exacerbate water loss in insect eggs. Elevated environmental temperatures and low relative humidity can increase water loss greatly. Insect eggs have adapted to these conditions by having an altered density or number of eggshell chorionic layers. The developing embryo also has different metabolic requirements as it progresses in development. As insect embryos develop into larvae, the metabolic rates and water loss rates increase [8]. These metabolic requirements peak shortly before the embryo hatches from the eggshell.

## **4. Insect egg management**

Eggs are undoubtedly the hardest insect life stage to kill with insecticides. Regardless of the application method, the tough eggshell that covers the egg prevents the entry of many insecticides, including water-based, oil-based, fumigants, and even some mechanical control methods.

An ovicide is a term used for an insecticide that specifically targets the egg stage. Smith and Salkeld [10] proposed three requirements for an ovicide to work: (1) the egg has to be in a location where it will be exposed so lethal concentrations of toxicant can reach it, (2) the egg has to be susceptible to the toxicant, and (3) a large enough proportion of eggs have to die from the toxicant in order for the treatment to be justified. The first requirement, exposure, is very important when considering insect eggs.

Agricultural pests, especially those that feed on plants, will commonly hide their eggs under leaves or may insert their eggs inside of the plant. Consideration of where the insect lays its egg in relation to an insecticide application is needed to guarantee an efficient treatment. The microclimate that the leaves create for the insect eggs is essential for their survival. Larger leaves, which absorb more sunlight, get much hotter than smaller leaves and can potentially kill eggs [11]. To limit sun exposure, insect eggs are often hidden underneath leaves and thus escape direct spray during an insecticidal treatment, resulting in an insect outbreak once those eggs hatch.

Systemic insecticides are often used for control of agricultural pests, but the use of these formulations presents a large problem for controlling the egg stage as far as exposure is concerned. These products are applied to the soil and then are taken up into the plant's xylem. Systemic products work well against immature and adult stages of sap-sucking pest insects, but these products do not work on eggs because the egg stage does not feed on the plant. Thus, the egg stage will survive and be a potential source for reinfestation.

If the eggs are not well hidden and the toxicant reaches the egg, the chemical still has to penetrate the eggshell and ultimately reach the embryo in order to be effective. Thus, the egg has to be susceptible to the toxicant. Variations in egg susceptibility could be due to the eggshell and differences in the chemical composition between eggs of different species [12] or could be due to enhanced resistance mechanisms of the embryo. Lastly, the toxicant must work well enough to kill a majority of the eggs in the population to be considered a viable option for control.

#### **4.1. Insecticides for insect egg control**

air layer below the outer chorion of the egg shell. The plastron acts as a physical gill that allows

Some terrestrial insect eggs that are oviposited in environments flooded with water may also have a plastron. In eggs with a large plastron membrane, only small parts of the plastron are permeable, limiting water loss. Terrestrial eggs typically do not have a plastron but do have a gas layer directly under the outer surface of the chorion and connected to the aeropyles. Aeropyles of terrestrial insects occasionally flooded by rainwater are located on the ends of respiratory horns. These horns extend above the water surface to allow for more efficient uptake of oxygen when the egg is surrounded by a layer of water [6]. These respiratory horns are essential to allow the insect to breathe during periods of heavy rain when an insect egg

The eggshell layers and elaborate respiratory systems of insect eggs are modified to conserve water for the insect egg. Terrestrial insect eggs are provided from their mother all of the water necessary for survival and development at the time of oviposition. Therefore, the embryos must conserve this limited amount of water during their development while also respiring. Water loss occurs across the chorion and is correlated to oxygen consumption requirements of the embryo. The more gas exchange that occurs, the more vulnerable the insect embryo is

The waxy layer in the insect eggshell most probably is the main barrier in the eggshell that prevents water loss. There was a significant increase in water loss in *Manduca sexta* eggs when the waxy layer was dissolved by organic solvents [8]. In addition to the waxy layer, the serosal membrane that envelopes the embryo has been shown to also protect insect eggs against desiccation [9]. RNAi technology has been used to prevent the development of the serosa in the beetle *Tribolium castaneum* [9]. Following RNAi treatment, the serosa-less eggs were subjected to a range of different humidities. Eggs exposed to the lowest humidities could not retain water and fewer eggs hatched compared to eggs that still had their serosal membrane

Environmental factors and embryonic development can exacerbate water loss in insect eggs. Elevated environmental temperatures and low relative humidity can increase water loss greatly. Insect eggs have adapted to these conditions by having an altered density or number of eggshell chorionic layers. The developing embryo also has different metabolic requirements as it progresses in development. As insect embryos develop into larvae, the metabolic rates and water loss rates increase [8]. These metabolic requirements peak shortly before the embryo

Eggs are undoubtedly the hardest insect life stage to kill with insecticides. Regardless of the application method, the tough eggshell that covers the egg prevents the entry of many insecticides, including water-based, oil-based, fumigants, and even some mechanical control

eggs to respire under water [6]

88 Insecticides Resistance

lacks the plastron respiratory system.

to water loss.

intact.

methods.

hatches from the eggshell.

**4. Insect egg management**

Normally, oil-based insecticides penetrate the insect eggshell more readily than water-based insecticides. The eggshell is comprised of a waxy component that allows the passage of oilbased products rather than water-based formulations. Early research has suggested that petroleum oils may also act by covering the aeropyles and causing egg mortality by limiting oxygen supply to the embryo [10]. Although most oils can be expected to penetrate the insect eggshell more easily, essential oils have been found to have difficulty penetrating eggs of the confused flour beetle and the Mediterranean flour moth compared to penetration through the cuticle of other life stages of the same insects, thus resulting in lower toxicity to eggs of these organisms [13]. Although oil-based products normally work better than water-based formu‐ lations in killing insect eggs, water has been shown to penetrate some insect eggshells. For instance, eggshell permeability to water has been demonstrated in the migratory locust, *Locusta migratoria migratoriodes* [14]. Empty, hatched migratory locust eggs were filled with water then placed into an osmotic solution, and water was observed both entering and leaving the eggshells. However, there is limited information regarding insect eggshells and their perme‐ ability to various chemicals. Consequently, the permeability of the insect eggshell to water, oil, or other chemical constituents of insecticides is not well established in the scientific literature.

Determining the mode of action of ovicidal insecticides is difficult. Formamidine insecticides, which have been evaluated against tobacco budworm (Lepidoptera: Noctuidae) eggs, were found to increase the levels of octopamine titers in eggs after treatment [15]. Formamidine insecticides have been shown to have a novel mode of action on insects by mimicking the actions of octopamine [16], which regulates insect behavior and energy metabolism. These results suggested that an increase in octopamine during embryogenesis could be playing a role in increased mortality of eggs treated with formamidine insecticides.

Paraoxon actively inhibits cholinesterase in *Pieris* eggs and was shown to prevent 100% of eggs from hatching [17]. Similarly, when house cricket eggs were exposed to carbamate insecticides, there was also a decrease in cholinesterase activity in eggs following exposure, but the insecticides did not prevent eggs from hatching [18].

*Triatoma infestans* eggs may be capable of detoxifying the organophosphate parathion with acetylcholinesterase enzymes [19]. The eggs produced elevated levels of acetylcholinesterase after being treated with parathion. The embryos were fully developed within the egg following treatment with parathion but never hatched from the eggshell. Therefore, the authors sug‐ gested that the embryos developed their nervous system during a later developmental stage and the parathion did not have an effect until the nervous system was fully developed.

All of these studies provide examples of insecticides permeating the eggshell and reaching the embryo, resulting in a physiological response to the insecticide. Unfortunately, most studies have only evaluated whether or not a particular insecticide has ovicidal action, thus lacking information on mode of action and penetration of the ovicide.

#### **4.2. Fumigation of insect eggs**

Fumigation has been found to be highly effective against eggs of several stored product pests. Eggs of four different species of common stored product pests, the almond moth (*Cadra cautella*), the Indian mealmoth (*Plodia interpunctella*), the lesser grain borer (*Rhyzopertha dominica*), and the red flour beetle (*T. castaneum),* have been evaluated to determine time, temperature, and pressures that were required for mortality by fumigation [20]. As tempera‐ tures increased and pressure decreased, time to mortality was reached in a shorter amount of time. Pressures above 100 mmHg and temperatures below 22.5°C were not practical to reach mortality because exposure times had to be increased drastically for the fumigant to cause egg mortality.

As is the case with liquid insecticides, studies have also shown that the egg stage is the most problematic life stage to kill with fumigants [12]. The main cause for low mortality is the impermeability of the eggshell. When eggs of *Schistocerca gregaria* were treated with sulfuryl fluoride, the gas was retained primarily in the proteinaceous portion of the eggshell instead of penetrating into the embryo [12]. In addition, egg age influences the efficacy of sulfuryl fluoride [20]. Eggs of the Mediterranean flour moth aged 1–2 days were the most tolerant to sulfuryl fluoride compared to younger and older eggs. Lower doses of sulfuryl fluoride were required to kill the Mediterranean flour moth as temperatures were increased from 15°C to 25°C.

Fumigation of stored product pests has been limited by EPA registration and regulations [20]. This concern has instigated investigations for alternative nonchemical control methods for treating stored-product insects. An alternative control method to fumigation for storedproduct pests that has been evaluated is the use of a vacuum system in storage bins to limit oxygen availability. However, eggs were also the most difficult life stage to kill using this method. This is not too surprising because an insect egg is very small and thus requires minute amounts of oxygen compared to the immature and adult stages of insects [20]. Therefore, extremely low levels of oxygen are required to create a hypoxic environment that will kill eggs.

#### **4.3. Transovarial transport of insecticides**

eggshells. However, there is limited information regarding insect eggshells and their perme‐ ability to various chemicals. Consequently, the permeability of the insect eggshell to water, oil, or other chemical constituents of insecticides is not well established in the scientific

Determining the mode of action of ovicidal insecticides is difficult. Formamidine insecticides, which have been evaluated against tobacco budworm (Lepidoptera: Noctuidae) eggs, were found to increase the levels of octopamine titers in eggs after treatment [15]. Formamidine insecticides have been shown to have a novel mode of action on insects by mimicking the actions of octopamine [16], which regulates insect behavior and energy metabolism. These results suggested that an increase in octopamine during embryogenesis could be playing a

Paraoxon actively inhibits cholinesterase in *Pieris* eggs and was shown to prevent 100% of eggs from hatching [17]. Similarly, when house cricket eggs were exposed to carbamate insecticides, there was also a decrease in cholinesterase activity in eggs following exposure, but the

*Triatoma infestans* eggs may be capable of detoxifying the organophosphate parathion with acetylcholinesterase enzymes [19]. The eggs produced elevated levels of acetylcholinesterase after being treated with parathion. The embryos were fully developed within the egg following treatment with parathion but never hatched from the eggshell. Therefore, the authors sug‐ gested that the embryos developed their nervous system during a later developmental stage and the parathion did not have an effect until the nervous system was fully developed.

All of these studies provide examples of insecticides permeating the eggshell and reaching the embryo, resulting in a physiological response to the insecticide. Unfortunately, most studies have only evaluated whether or not a particular insecticide has ovicidal action, thus lacking

Fumigation has been found to be highly effective against eggs of several stored product pests. Eggs of four different species of common stored product pests, the almond moth (*Cadra cautella*), the Indian mealmoth (*Plodia interpunctella*), the lesser grain borer (*Rhyzopertha dominica*), and the red flour beetle (*T. castaneum),* have been evaluated to determine time, temperature, and pressures that were required for mortality by fumigation [20]. As tempera‐ tures increased and pressure decreased, time to mortality was reached in a shorter amount of time. Pressures above 100 mmHg and temperatures below 22.5°C were not practical to reach mortality because exposure times had to be increased drastically for the fumigant to cause egg

As is the case with liquid insecticides, studies have also shown that the egg stage is the most problematic life stage to kill with fumigants [12]. The main cause for low mortality is the impermeability of the eggshell. When eggs of *Schistocerca gregaria* were treated with sulfuryl fluoride, the gas was retained primarily in the proteinaceous portion of the eggshell instead of penetrating into the embryo [12]. In addition, egg age influences the efficacy of sulfuryl

role in increased mortality of eggs treated with formamidine insecticides.

insecticides did not prevent eggs from hatching [18].

information on mode of action and penetration of the ovicide.

**4.2. Fumigation of insect eggs**

mortality.

literature.

90 Insecticides Resistance

The transovarial transport of insect growth regulators from mother to offspring has been shown to cause a considerable reduction in egg hatch and viability. Insect growth regulators affect the development and occasionally the reproduction of insects. There are two main types of insect growth regulators: juvenile hormone analogs and chitin synthesis inhibitors. Juvenile hormone analogs (JHAs) mimic the natural juvenile hormone present in insects and can cause multiple physiological and morphological problems. Chitin synthesis inhibitors (CSIs), as the name suggests, inhibit the proper formation of chitin between insect molts. CSIs can cause insects to have malformed, thinner cuticles and ruptured intestines. Many of the symptoms will lead to eventual death of the insect if they do not die at the time of molt.

The transovarial transport of insect growth regulators from adult female insects to eggs have been evaluated in several insect species. Different insect species have varying tolerances and responses to pyriproxifen. For example, none of the eggs of gravid whitefly, *Bemisia tabaci*, females treated with the juvenile hormone analog pyriproxifen hatched [21]. Alternatively, when pyriproxifen was applied to female adults of the common green lacewing, *Chrysoperla carnea*, it had little effect on preventing their eggs from hatching [22]. Transovarial transport has also been documented with diflubenzuron, a chitin synthesis inhibitor. Treatment of adult female *C. carnea* with diflubenzuron resulted in 100% mortality of eggs at the highest tested dose.

The penetration of insecticides into the female's ovaries and elsewhere in the female body has been evaluated by using [14C]-labeled isotopes [22]. Most of the pyriproxifen in the female adult *C. carnea* was excreted within a couple of days, so it was probably not present before oviposition [22]. The use of the radio-labeled isotopes showed that diflubenzuron, unlike pyriproxifen, was absorbed more slowly and was retained within the female's body, which explains the high levels of toxicity to eggs with this insecticide.

#### **4.4. Fungi as biological control agents for eggs**

Entomopathogenic fungi have been used on a variety of insect species as a biological control agent, but the egg stage has not been found to be highly susceptible to fungal pathogens in several cases. Eggs of the greenhouse whitefly, *Trialeurodes vaporariorum*, were found to be nonsusceptible to the fungus *Aschersonia aleyrodis* [23]. No fungal spores or discoloration of the eggs was observed, and there were no differences in mortality between eggs that were treated with the fungus compared to eggs that had not been treated. However, the fungal spores were persistent for several days, so when the first instar larvae emerged from the egg, they became infected with the fungus.

Five different fungi, (*Beauveria bassiana, Metarhizium anisopliae, Metarhizium flavoviride, Paeci‐ lomyces farinosus*, and *Paecilomyces fumosoroseus*) were tested against eggs of the curculionidae beetles, *Otiorhynchus sulcatus* and *Sitona lineatus* [24]. *S. lineatus* eggs were much more tolerant of the fungal pathogens compared to *O. sulcatus* eggs. *O. sulcatus* eggs were found to be susceptible to all fungi except for *B. bassiana*. Only one fungus, *M. flavoviride*, was found to be moderately effective against *S. lineatus* eggs, causing 32% egg mortality. No other fungal treatments resulted in egg mortality to *S. lineatus* eggs.
