Comparision of Antioxidant Activity of Cow and Goat Milk During Fermentation with *Lactobacillus acidophilus* LA-5

*Jessica Dayara Álvarez-Rosales, César Ozuna, Rubén Salcedo-Hernández and Gabriela Rodríguez-Hernández*


*Article* **Effects of Temperature on** *Anoplophora chinensis* **(Coleoptera: Cerambycidae) Adult Survival, Reproduction, and Egg Hatch**

**Melody A. Keena \* , Paul M. Moore and Gregg Bradford**

Northern Research Station, Northeastern Center for Forest Health Research, USDA Forest Service, Hamden, CT 06514, USA<EMAIL_ADDRESS>(P.M.M<EMAIL_ADDRESS>(G.B.) **\*** Correspondence<EMAIL_ADDRESS>
**Abstract:** *Anoplophora chinensis* (Forster) is an invasive species that can damage many tree species in orchard, urban, and forested habitats. Adult survival, reproduction, and egg hatch of *A. chinensis* from Italy and China are evaluated at eight constant temperatures (5, 10, 15, 20, 25, 30, 35, and 40 ◦C) under laboratory conditions. The estimated *Tmax* for longevity was 42 and 33 ◦C for females and 42 and 39 ◦C for males from China and Italy, respectively. The estimated *Tmax*, *Tmin*, and optimum temperature for fecundity were 35, 9, and 29 ◦C, respectively. Females laid eggs at 15–30 ◦C and eggs hatched at 15–35 ◦C. Days to first oviposition increased exponentially from 13 days at 30 ◦C to >300 days near 10 ◦C. The estimated *Tmin* for egg hatch was 13 ◦C, the *Tmax* at 38 ◦C, and the optimum 29 ◦C. Percentage hatch was estimated to be highest at 26 ◦C and have a *Tmax* of 31 ◦C and *Tmin* of 10 ◦C. These results indicate that summer temperatures over a wide range of latitudes should support beetle survival and reproduction, but at temperatures ≥35 ◦C, oviposition ceases, and adult survivorship declines. In addition, females may survive into the fall, but lay fewer eggs that may not hatch. These responses of *A. chinensis* to temperature can be used for developing phenological models to predict the timing of stages for management or eradication efforts.

**Keywords:** *Anoplophora chinensis*; temperature; survival; reproduction; fecundity

### **1. Introduction**

Temperature is an important abiotic factor that influences insect life-history processes, such as survival, development, and reproduction. Understanding how insects respond to temperature makes it possible to estimate potential geographic ranges and develop phenology models to predict the timing of stages that are critical to deploying survey and management options. It is particularly important to develop these predictive tools for species like *Anoplophora chinensis* (Forster) (Coleoptera: Cerambycidae) that become invasive after being transported, either in live plants or solid wood packaging materials, through international trade routes. The native range of *A. chinensis* largely overlaps that of its congeneric, *Anoplophora glabripennis* (Motschulsky), but it has been recorded from warmer areas of southern Asia and is not found in some of the more northern parts of China and far east Russia [1]. *Anoplophora chinensis* is widely distributed in China, Japan, and Korea, and occasionally found in Vietnam, Malaysia, Indonesia, and the Philippines [2,3]. It is a polyphagous xylophage that attacks 73 different deciduous tree genera (more than *A. glabripennis*) and also is reported to attack some coniferous trees in the genera *Cryptomena* and *Pinus* [4]. The beetle is the most important wood infesting citrus pest in Japan and China, and also causes major damage to *Casuarina equisetifolia* L. shelterbelts in China [5–7]. In its invaded range, *A. chinensis* prefers trees in the genera *Acer*, *Betula*, and *Corylus* but has completed development on 24 genera and partial development on another five genera of trees [3,8]. Females lay their eggs in sites they chew in the bark of the lower bole or exposed roots of the tree, and the early instars feed under the bark, while later instars feed in the xylem [3]. The larvae cause damage to the vascular system of the tree and create structural

**Citation:** Keena, M.A.; Moore, P.M.; Bradford, G. Effects of Temperature on *Anoplophora chinensis* (Coleoptera: Cerambycidae) Adult Survival, Reproduction, and Egg Hatch. *Forests* **2021**, *12*, 432. https://doi.org/ 10.3390/f12040432

Academic Editors: Salvatore Moricca and Tiziana Panzavolta

Received: 26 February 2021 Accepted: 1 April 2021 Published: 3 April 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

weaknesses that can lead to the death of the tree. Adults are present from the late spring through the summer and into the early fall, depending on the local climatic conditions.

A recent revision of *Anoplophora* considered *Anoplophora malasiaca* (Thomson) to be a synonym of *A. chinensis* based on shared morphological traits of the reproductive system [2]. There is, however, still controversy over this synonymy, since they can be differentiated by whether or not two white spots are present on the pronotum and by their mitochondrial COI region haplotypes [9,10]. The first invasive population of *A. chinensis* was found in the Lombardy region of Italy in 2000, but in the last 20 years, additional infestations have been found in Italy, France, the Netherlands, Croatia, and Turkey [11]. The infestations in the Netherlands and Croatia have been traced to live *Acer palmatum* Thunberg imported from China, while the one in France started with beetles that emerged from bonsais imported from Asia [11,12]. A find outside a greenhouse in Tukwila, Washington, United States in 2001 also came from imported bonsais [13]. Using the mitochondrial DNA haplotypes, the Italian infestations from the Lazio and Lombardy regions were found to be similar to Japanese populations, while those from Tuscany were closer to Chinese populations [14]. Therefore, the invasive populations may originate from areas where the two original species are found and could be different biologically in their responses to temperature and other traits. Since all invasive infestations or potential ones (Washington State, US) have resulted in the establishment of eradication efforts, it is important to know if there are differences between these original species. For simplicity, *A. malasiaca* will be referred to as "*A. chinensis* from Japan".

There is some information on the temperature effects on the life-history of *A. chinensis* adults and eggs. At natural temperatures on citrus (twigs for food and bolts for oviposition) adults from a Japanese population of *A. chinensis* survived 77.6 ± 20.3 days, maturation fed for 9.8 ± 1.4 days, and females laid 193.8 ± 65.2 eggs (peaked at 30 days post eclosion and 90% of the eggs hatched) [7]. Another study using Japanese adults found differences in maturation feeding (feeding required to reach sexual maturity), longevity, and fecundity at 24 ◦C when different hosts were used [15]. Fecundity ranged from 60–140 eggs when another group of Japanese females was offered *Acer*, *Citrus*, and *Platanus* bolts to lay on in outdoor cages [10]. When Chinese *A. chinensis* females were fed on *Melia azedarach* L., on average, they laid 176 eggs and lived 92 days (males 103 days), but they only laid 56 eggs and lived 62 days (males 63 days) when fed *Casuarina* sp. [1]. Finally, it was estimated that a total of 184 degree-days is required for 50% of Japanese *A. chinensis* eggs to hatch when using the estimated *Tmin* of 6.7 ◦C [7].

In this study, we determined the response of *A. chinensis* adults and eggs to a range of constant temperatures. Then the mathematical relationships between temperature and adult survival, reproduction, and egg hatch were developed for *A. chinensis* to facilitate their use in developing phenology models and to compare them to what has been previously reported for *A. glabripennis*. Finally, the implications for management and eradication programs are discussed.

### **2. Materials and Methods**

### *2.1. Populations and Temperatures*

The populations used in this study were established from larvae that were shipped on 26 February 2018, from the USDA Agricultural Research Service (ARS), European Biological Control Laboratory in Montferrier-sur-Lez, France, under permit to the USDA Forest Service quarantine facility in Ansonia, CT. Fifty larvae from the fifteenth ARS laboratory generation of an invasive *A. chinensis* population from Lombardy, Italy (41.58◦ N and 87.42◦ W) were used to start the USDA Forest Service (FS) colony. The Italian colony was initiated by ARS in 2002 and augmented each year with freshly collected adults. The Italian colony was determined to have originated from Japan based on molecular tests. Individuals used in this study were from the sixteenth or seventeenth Italian laboratory generations.

Fifty larvae from the fifth ARS generation of a native *A. chinensis* population from Yancun, Guangdong Province, China colony (40.45◦ N and 73.45◦ W) were used to start

the FS colony. The Chinese population was started by ARS in 2012 with 180 larvae from China. Individuals used in this study were from the sixth or seventh laboratory generations. Voucher specimens of both populations were deposited at the Entomology Division, Yale Peabody Museum of Natural History, New Haven, CT.

Eight constant temperatures were used for these studies: 5, 10, 15, 20, 25, 30, 35, and 40 ◦C. Temperature fluctuations in the environmental chambers rarely exceeded 1 degree above or below the set value, and a photoperiod of 16:8 (L:D) h was used. Humidity was passively maintained by placing open reservoirs of water (32 × 28 × 14 cm3 ) in the bottom of the chambers or by humidifiers that were part of the chamber (15, 20 and 25 ◦C). The humidity in the chambers averaged 80 ± 10%, 85 ± 15%, 70 ± 10%, 80 ± 5%, 60 ± 5%, 45 ± 5%, 68 ± 4%, and 55 ± 10% RH at 5, 10, 15, 20, 25, 30, 35, and 40 ◦C, respectively. Actual humidity within the rearing containers was likely higher than in the chamber, due to the presence of fresh host material.

### *2.2. Mating Adults and Obtaining Eggs*

Adults used in these studies came from larvae reared on an artificial diet, either the *A. glabripennis* diet designated "AG2" [16] or a drier *A. chinensis* diet that was a modification of a host-free diet developed for *A. glabripennis* [17,18]. Pre-pupae were placed in 50 mL plastic centrifuge tubes (with two pinholes in the lid and a piece of damp paper towel) and held in the dark at 25 ◦C until pupae formed, and the adults emerged. Callow adults were held for four more days under the same conditions as the pre-pupae/pupae to allow the cuticle to fully sclerotize before they were sexed, weighed, and assigned to a temperature regime. Chinese adults (15 females and 16–18 males) were initially assigned evenly to the 15, 20, 25, and 30 ◦C temperature regimes, and then ten of each sex were assigned evenly to the 10, 35, and 40 ◦C temperature regimes. Virgin females in the 15–35 ◦C temperature regimes were held individually in 3.8-L glass jars with *Acer rubrum* L. (red maple) twigs (3–7 mm diameter with leaves removed) as a food source and *A. rubrum* bolts (3–7 cm diameter and 22 cm long) with both ends waxed as an oviposition substrate. A virgin male was added to the female's jar 0–11 days later (i.e., as soon as a female became available at the same temperature). Males that were waiting for a mate were held at their assigned temperatures in 950 mL glass jars with fresh twigs as a food source. All adults held at 10 and 40 ◦C were held individually in 950 mL glass jars with fresh twigs as a food source. After twelve weeks at 10 ◦C (presumably enough time for the females to maturation feed), the three males that were still alive were placed with females in the larger glass jars to see if the females would chew pits or lay eggs. Twigs and bolts were cut twice a month and were stored at 4 ◦C and ≥80% RH until used in the weekly food and bolt changes. Folded paper towels were placed in the bottoms of the jars to collect frass and excess moisture. Two holes (1–2 mm diameter) in the plastic jar lids allowed airflow.

The oviposition bolts were replaced twice a week (30–35 ◦C), weekly (15–25 ◦C), or monthly (10 ◦C) until a female died. Oviposition bolts held at 15–35 ◦C were checked daily for oviposition sites. The date of each new oviposition pit chewed in the bark was recorded, and the pit marked to distinguish the day it appeared. When the oviposition bolts were removed from the jars, all pits chewed in the bark, including both pits that obviously contained eggs and those that did not, were counted, and the bolt diameter was measured.

The bark was stripped off many of the daily marked bolts the day they were removed from the jar to obtain the eggs for the egg hatch part of the study. After the bark was stripped off, the eggs were carefully removed from the bolt by lifting a thin strip of wood with the egg on it to prevent breaking the eggshell. Eggs attached to the small pieces of wood were placed individually in wells of a 24-well tissue culture plate that was held in a water box at the temperature at which the egg was laid or to which the egg was moved. Ten eggs maximum, or half of the eggs at minimum, that were laid by females at 15 ◦C were kept at that temperature. For females held at other temperatures, a minimum of 10 eggs was held at the oviposition temperature, and the others were moved as needed.

To minimize temperature shifts for eggs that had already begun developing, one-dayold eggs were only moved to temperatures that were ±5 ◦C of the laying temperature, but eggs laid the same day were moved to other temperatures as needed. Eggs laid more than one day before the bolt was removed from the mating jar were held at the oviposition temperature. Eggs laid by Chinese females held at 25 or 30 ◦C were moved to any temperature, while eggs laid at 15 ◦C were only moved to 20–30 ◦C and eggs laid at 20 ◦C were only moved to 10–35 ◦C. This was because there were fewer eggs laid at the cooler temperatures. Two to ten eggs from a Chinese female were held at each temperature 15–30 ◦C, and 1–3 eggs were held at each temperature 5–10 ◦C and 35–40 ◦C.

Once enough eggs were obtained from each mating pair for the egg hatch part of the study, the rest of the bolts from each female were held at 25 or 30 ◦C for 7–10 days to allow the eggs to hatch before stripping the bark off the bolt. This provided a better measure of the percentage of the eggs that were fertile, since removing the eggs could damage or kill them. Eggs were checked daily for 4, 4, 4, 6, 9, 12, 30, and 30 weeks at 40, 35, 30, 25, 20, 15, 10, and 5 ◦C, respectively. These time periods were chosen to ensure that all eggs that could hatch at a given temperature did. Eggs that had not hatched after the given time period at 5–20 ◦C were moved to 25 ◦C and observed for an additional four weeks.

Adults from the invasive Italian population were more limited in numbers, so only 8–10 females and 11–14 males were assigned on a rotating basis to the 15, 20, 25, and 30 ◦C temperature regimes. All the same feeding and holding methods were used as detailed above for the Chinese population adults and eggs. Since Italian females also laid fewer eggs, their eggs were only held at 15–30 ◦C. Eggs laid at these temperatures were moved using the same scheme as detailed for the Chinese eggs.

### *2.3. Adult Survival and Reproduction*

Mating pairs and single adults were checked daily for mortality, with the only exception that single adults at 10 ◦C were checked three times a week. When the female in the pair died, the male was moved to a 950 mL glass jar and maintained until death. When the male in the pair died, the female was maintained in the mating jar until death. For each temperature at which females laid eggs, the mean number of eggs laid per week per live female during that week was calculated. When the female died, she was dissected to determine if she had any fully formed eggs in her ovarioles that she had not oviposited. The following dependent variables were analyzed in PROC GLIMMIX [19]: Female and male weight at four days, female and male longevity, days to the first egg, fecundity, maximum weekly oviposition, a week at which maximum oviposition occurred, mean weekly oviposition pits per female, mature unlaid eggs at female death, and percentage hatch on bolts held at 25–30 ◦C. A gamma distribution with a log link was used for all the variables except the percentage hatch for which a beta distribution with a logit link was used and the unlaid eggs in the female at death for which a Poisson distribution with a log link was used. The two populations were analyzed separately for each variable. The model used temperature, population (Italy or China), and the interaction between the two as fixed effects. Differences among means were determined using the Tukey–Kramer test with α = 0.05 [19].

The relationship between longevity and temperature was separately fit for each sex and population combination to a linear model (*y* = *bx* + *a*), and the upper threshold (*Tmax*) was calculated to be the point at which the line crossed the *x*-axis. The relationship between fecundity and temperature for the Chinese population was fit using the following:

$$y = \begin{cases} \
\,\,\,at(t - T\max)(T\min - t)^{\frac{1}{\theta}}\,\,\,\,\text{for}\,\,T\max \le t \le T\min\\ \text{otherwise 0} \end{cases} \tag{1}$$

where *y* = fecundity, *t* = temperature ◦C are the known values, and the others are estimated. The a is an empirical constant, b modulates the slopes, and *Tmax* and *Tmin* are the upper and lower temperature thresholds, respectively [20]. Nonlinear convergence for the model

was based on the Marquardt method (PROC NLIN [19]). The optimum temperature for Equation (1) was calculated by equating the first derivative to zero and solving for t.

The relationship between temperature (*t*) and *a* number of days from female emergence to first oviposition was fit using the following:

$$y = at^b\tag{2}$$

where *a* and *b* are empirical constants, and nonlinear convergence was based on the Marquardt method (PROC NLIN [19]). The percentage of females that oviposited and produced progeny was also calculated for each temperature and population combination.

### *2.4. Egg Hatch*

Two dependent variables were analyzed using PROC GLIMMIX [19]: Days to hatch and percent hatch on bolts held at 25–30 ◦C. The model treated temperature, population, and temperature by population as fixed effects, whereas maternal family was treated as a random effect. Differences among means were determined using the Tukey–Kramer test with α = 0.05 [19]. A gamma distribution with a log link was used for days to hatch, and a beta distribution with a logit link was used for the percentage hatch on bolts. The percentage hatch of eggs laid at or moved to various temperatures after being removed from under the bark was also calculated.

To describe the relationship between temperature and rate of hatch for eggs from the combined populations, Equation (1) was altered by changing *y* = 1/days to hatch, and the curve was fit using the Marquardt convergence method (PROC NLIN [19]). The cumulative distributions of time to egg hatch at 15, 20, 25, and 30 ◦C and degree days to egg hatch for all eggs were calculated using the estimated *Tmin* from Equation (1). The cumulative proportion of eggs hatching (*P*) over accumulated degree-days (*DD*) was described using a Gompertz function:

$$P = \exp\left(-\exp\left(-bDD + a\right)\right) \tag{3}$$

in which *a* and *b* are the lag and the rate of increase, respectively ([21], PROC NLIN and Marquardt convergence method, [19]).

### **3. Results**

### *3.1. Adult Survival and Reproduction*

At the initiation of the temperature treatments, adult females from China (186.5 ± 28.9 mg) were significantly heavier than females from Italy (145.3 ± 25.0 mg) (*F* = 39.96, df = 1, 93, *p* < 0.0001). There was also a significant difference (*F* = 40.21, df = 1, 139, *p* < 0.0001) in the average mass of males from the Chinese population (127.5 ± 22.8 mg) and the Italian population (105.1 ± 21.0 mg). The mass of the beetles held at the different temperatures for either sex did not differ (females: *F* = 0.46, df = 6, 93, *p* = 0.8351; males: *F* = 1.17, df = 6, 139, *p* = 0.3274).

Both male and female longevity increased with decreasing temperature and Italian individuals of both sexes tended to survive a shorter time than Chinese individuals (Table 1). Temperature and population both had significant effects on both female (temperature: *F* = 126.6, df = 6, 113, *p* < 0.0001; population: *F* = 39.3, df = 1, 113, *p* < 0.0001) and male longevity (temperature: *F* = 127.8, df = 6, 139, *p* < 0.0001; population: *F* = 60.2, df = 1, 139, *p* < 0.0001), but the interaction between the two did not (Table 1). Female and male longevity fit a linear model, but variation increased at the lower temperatures so that the confidence of the fit (adjusted *R 2* ) was lower, especially for the Italian population (Figure 1, Table 2). At 10 ◦C the Chinese population appeared to have two biotypes, one that was dominated by males that survived about 100 days and another that was dominated by females that survived about 375 days. The estimated *Tmin* for each sex and population combination for longevity and the parameters for the fitted models are given in Table 2.

*Forests* **2021**, *12*, 432

**Table 1.** Effects of temperature on *Anoplophora chinensis* adult survival and reproduction ((mean ± SE (n) or percentage (n)). Means were compared between each temperature and population combination for each parameter.

Parameter values are means followed by standard errors. In the table and the models, *TMax* = upper temperature threshold and *TMin* = lower temperature threshold. The temperature range used did not always include all the data points, since some were above or below the thresholds based on preliminary plots. a model *y* = *ax* + *b*. b model *y* = ( at(*t* − *Tmax*)(*Tmin* − *t*) 1 *b* for *Tmax* ≤ *t* ≤ *Tmin* otherwise 0 , where *t* is the

temperature (◦C). c model *y* = *atb*

.

**Figure 1.** Relationship between temperature and longevity for male and female *A. chinensis* from China and Italy. Observed male data are open triangles, observed female data are open circles, predicted male dashed line, and predicted female solid line. **Figure 1.** Relationship between temperature and longevity for male and female *A. chinensis* from China and Italy. Observed male data are open triangles, observed female data are open circles, predicted male dashed line, and predicted female solid line.

Once Chinese females started laying eggs, they generally continued laying eggs until about five weeks before they died, while Italian females stopped laying at about two weeks before death at 30 ◦C. The time to first oviposition increased with decreasing temperature with the maximum being 12 weeks at 15 ◦C (Figure 2). The fecundity of *A. chinensis* from China peaked at 30 ◦C and declined with both increasing and decreasing temperatures, while the fecundity of females from Italy did not vary much over the 15–30 ◦C temperature range (Table 1, Figure 2). This effect of temperature on fecundity was significant (*F* = 2.93, df = 3, 69, *p* = 0.0394) and so was the effect of population (*F* = 31.47, df = 1, 69, *p* < 0.0001), but the interaction between the two was not (Table 1). Both the percentage of Italian females that laid and produced progeny were also lower than that for Chinese females, further reducing their total reproduction over the 15–30 ◦C temperature range (Table 1). Equation (1) described the relationship between the mean fecundity of Chinese females and temperature reasonably well based on the adjusted *R 2* value (Table 2, Figure 3A). No curve was fit for the Italian population because fecundity varied little over the range of temperatures where oviposition occurred.

There were significant effects of temperature (*F* = 8.33, df = 3, 69, *p* < 0.0001) and population (*F* = 23.6, df = 1, 69, *p* < 0.0001) on mean maximum weekly oviposition, peaking at 26 eggs per week at 30 ◦C and declining below that temperature for Chinese females and remaining constant at about eight eggs per week for Italian females (Table 1). The maximum weekly oviposition recorded for any female was 12, 22, 26, and 35 eggs for Chinese females and 12, 15, 17, and 19 eggs for Italian females at 15, 20, 25, and 30 ◦C, respectively. The week that the maximum weekly oviposition occurred was significantly affected by temperature (*F* = 40.32, df = 3, 69, *p* < 0.0001), but not population (*F* = 43.77, df = 1, 69, *p* < 0.0001); oviposition peaked sooner at higher temperatures than at lower ones (Table 1). The number of days to first oviposition was significantly affected by the interaction between temperature and population (*F* = 5.26, df = 3, 69, *p* = 0.0025), generally

declining with increasing temperature (Table 1). Equation (2) described the relationship between temperature and number of days to first oviposition well with an adjusted *R* 2 of 0.88 (Figure 3B). The average number of oviposition pits chewed weekly per female was significantly affected by both temperature (*F* = 23.03, df = 4, 80, *p* < 0.0001) and population (*F* = 40.09, df = 1, 80, *p* < 0.0001), peaking at 25 ◦C and declining both above (Chinese population) and below those temperatures (Table 1). *Forests* **2021**, *12*, x FOR PEER REVIEW 11 of 18

**Figure 2.** Mean (±SE) weekly oviposition and female survival for female *A. chinensis* from China (Left) and Italy (Right) held at four constant temperatures. **Figure 2.** Mean (±SE) weekly oviposition and female survival for female *A. chinensis* from China (Left) and Italy (Right) held at four constant temperatures.

There were significant effects of temperature (*F* = 8.33, df = 3, 69, *p* < 0.0001) and population (*F* = 23.6, df = 1, 69, *p* < 0.0001) on mean maximum weekly oviposition, peaking at 26 eggs per week at 30 °C and declining below that temperature for Chinese females and remaining constant at about eight eggs per week for Italian females (Table 1). The maximum weekly oviposition recorded for any female was 12, 22, 26, and 35 eggs for Chinese females and 12, 15, 17, and 19 eggs for Italian females at 15, 20, 25, and 30 °C, respectively. The week that the maximum weekly oviposition occurred was significantly affected by The interaction between temperature and the population the female came from significantly (*F* = 3.81, df = 3, 113, *p* = 0.0121) affected the number of mature unlaid eggs found in the female at death. At temperatures between 15 and 30 ◦C there was no difference in the number of unlaid eggs for Italian females (Table 1). Chinese females had no unlaid eggs at 10 and 40 ◦C, and only one female had a single matured egg in her ovarioles at death at 35 ◦C. Chinese females at 15 ◦C had fewer unlaid eggs than those at 30 ◦C. The maximum number of unlaid mature eggs found in females from both populations was eight eggs.

### temperature (*F* = 40.32, df = 3, 69, *p* < 0.0001), but not population (*F* = 43.77, df = 1, 69, *p* < *3.2. Egg Hatch*

0.0001); oviposition peaked sooner at higher temperatures than at lower ones (Table 1). The number of days to first oviposition was significantly affected by the interaction between temperature and population (*F* = 5.26, df = 3, 69, *p* = 0.0025),generally declining with increasing temperature (Table 1). Equation (2) described the relationship between temperature and number of days to first oviposition well with an adjusted *R*2 of 0.88 (Figure 3B). The average number of oviposition pits chewed weekly per female was significantly affected by both temperature (*F* = 23.03, df = 4, 80, *p* < 0.0001) and population (*F* = 40.09, df = 1, 80, *p* < 0.0001), peaking at 25 °C and declining both above (Chinese population) and below those temperatures (Table 1). Temperature had a significant effect (*F* = 392.34, df = 4, 897, *p* < 0.0001) on number of days to egg hatch, but population did not (*F* = 0.13, df = 1, 897, *p* = 0.7202). The number of days to egg hatch for the populations combined was lowest at 30 ◦C and increased both above and below that temperature (Table 3). The relationship between hatch rate (1/d) and temperature was fit to Equation (1) with good confidence (*R* 2 = 0.529, Table 2, Figure 4B). The *Tmin* and *Tmax* for hatch rate appear to be good estimates, since no hatch occurred at 5, 10, or 40 ◦C, although only eggs from the Chinese population were used at these temperatures (Table 3).

**Time to Egg**

**% Hatch of Eggs Removed from Under the Bark**

*Forests* **2021**, *12*, x FOR PEER REVIEW 13 of 18

**Table 3.** Effects of temperature on *Anoplophora chinensis* egg hatch (mean ± SE (n)).

**Figure 3.** Relationships between temperature and mean fecundity (**A**) and time to oviposition (**B**) for *A. chinensis* from China (solid squares) and Italy (open circles). The solid line ingraph A is for only the Chinese population, and ingraph B it is for the two populations combined. **Figure 3.** Relationships between temperature and mean fecundity (**A**) and time to oviposition (**B**) for *A. chinensis* from China (solid squares) and Italy (open circles). The solid line in graph A is for only the Chinese population, and in graph B it is for the two populations combined. hatch of Chinese eggs on bolts was fit with high confidence (*R*2 = 0.992) by Equation (1), but no curve was fit for the Italian egg data, because the maximum likelihood estimator did not converge (Figure 4A, Table 2).

10.5 ± 0.1cd (770)

> 60.5 (1138)

52.9 (155) 9.9 ± 0.2d (96)

> 27.3 (333)

> > 7.4

12.3 ± 0.9c (6)

> 5.5 (109)

(68) NA NA

0.0 (51)

high confidence (*R*2 = 0.994; Figure 5B). Degree-day accumulations of 125.5 (95% CI 125.3– 125.6) and 165.6 (95% CI 164.4–167.0), are predicted for 50% and 90% of the eggs to hatch, respectively (Figure 5B, 0.5 and 0.9 values). **Figure 4.** Relationships between temperature and mean percentage egg hatch (**A**) and mean hatch rate (1/d) (**B**) for *A. chinensis*. Ingraph A, observed points are solid squares (Chinese) and open circles (Italian), and the predicted line is only for the Chinese population. Results are pooled across populations ingraph B (solid squares are mean observed points). **Figure 4.** Relationships between temperature and mean percentage egg hatch (**A**) and mean hatch rate (1/d) (**B**) for *A. chinensis*. In graph A, observed points are solid squares (Chinese) and open circles (Italian), and the predicted line is only for the Chinese population. Results are pooled across populations in graph B (solid squares are mean observed points).

**Table 3.** Effects of temperature on *Anoplophora chinensis* egg hatch (mean ± SE (n)).

Time to egg hatch values followed by the same letter are not significantly different (*F* = 392.34, df = 4, 897, *p* < 0.0001) based on the Tukey–Kramer test with α = 0.05 [19]. The data for the Chinese and Italian populations were pooled for time to hatch. NA = parameter not applicable, since no eggs were held or hatched at these temperatures. Some eggs laid the day the bolt was removed, or the previous day, were moved to other temperatures and held for hatch. Other eggs were held at the oviposition temperature.

The cumulative proportions of the number of days to hatch at 15 to 35 ◦C (populations pooled) is shown in Figure 5A. The first hatch occurred at 31, 9, 5, 5, and 11 days and the last at 50, 30, 19, 16, and 15 days for eggs held at 15, 20, 25, 30, and 35 ◦C, respectively. The cumulative proportion versus accumulated degree-days was fit to Equation (3) with high confidence (*R* 2 = 0.994; Figure 5B). Degree-day accumulations of 125.5 (95% CI 125.3–125.6) and 165.6 (95% CI 164.4–167.0), are predicted for 50% and 90% of the eggs to hatch, respectively (Figure 5B, 0.5 and 0.9 values). *Forests* **2021**, *12*, x FOR PEER REVIEW 14 of 18

**Figure 5.** Observed cumulative frequency distributions for *A. chinensis* egg hatch times (d) at five constant temperatures (°C) (**A**). Cumulative proportion of *A. chinensis* eggs hatching over accumulated degree-days (**B**). The solid line in the bottomgraph represents the fit to the equation: Predicted proportion hatching = exp (–exp (0.0469 (±0.00139) X accumulated degree-days + 5.5186 (±0.1683))); adjusted *R*2 = 0.994. **Figure 5.** Observed cumulative frequency distributions for *A. chinensis* egg hatch times (d) at five constant temperatures (◦C) (**A**). Cumulative proportion of *A. chinensis* eggs hatching over accumulated degree-days (**B**). The solid line in the bottom graph represents the fit to the equation: Predicted proportion hatching = exp (–exp (0.0469 (±0.00139) X accumulated degree-days + 5.5186 (±0.1683))); adjusted *R* 2 = 0.994.

**4. Discussion** The observed adult activity over the range of temperatures assessed was comparable The percentage hatch of eggs laid at or moved to temperatures for each population is given in Table 3. Because the percentage hatch for eggs removed from under the bark

temperatures. At 10 °C Chinese adults moved very slowly, fed little, and did not chew oviposition sites. At 30–35 °C adult behavior increased, and they would attempt to walk or fly away when their twigs and bolts were changed. At 35 °C, Chinese females did not oviposit even though they chewed some oviposition pits. Chinese adults held at 40 °C were more lethargic, fed little, and did not live long. The attempts to move away from the higher temperatures fit with the observations that *A.glabripennis* seeks shaded areas to rest

Adult longevity declined with the increase in activity at higher temperatures. This is likely due to increased metabolism at higher temperatures which has been found to reduce the life span in insects [25]. The longevity at 20–25 °C on *A. rubrum* for both sexes from the Chinese population is comparable to that recorded on *Melia azedarach* (91.8 days female and 103.0 days male) and higher than recorded on *Casuarina* sp. (61.6 days female and 63.0 days male) at room temperature in China [1]. Conversely, both sexes of the Italian population on *A. rubrum* at 25 °C lived for a shorter time than did individuals from a Japanese population at 24 °C on either *Citrus unshiu* (Yu.Tanaka ex Swingle) or *Salix schwerinii* E. Wolf [15]. This highlights the effects of host plants on longevity and suggests that caution should be used when applying predictions solely based on a single host in the laboratory. Female *A. chinensis* from both populations lived longer than *A.glabripennis* females at temperatures ≤15 °C, but comparable lengths of time at higher temperatures [23].

when the temperature is >29 °C in China [24].

seemed low, the percentage hatch for eggs laid at different temperatures then held on bolts at 25–30 ◦C was used for developing the relationship between percentage hatch (as a measure of fertility) and temperature. Percentage hatch on bolts was significantly affected by temperature (*F* = 5.85, df = 3, 63, *p* = 0.0014), but not population or the intersection between the two (Table 1). Percentage hatch was highest at 25 ◦C for the Chinese population and 20 ◦C for the Italian population and declined as temperature increased or decreased (Table 1, Figure 4A). The relationship between temperature and mean percentage hatch of Chinese eggs on bolts was fit with high confidence (*R* 2 = 0.992) by Equation (1), but no curve was fit for the Italian egg data, because the maximum likelihood estimator did not converge (Figure 4A, Table 2).

### **4. Discussion**

The observed adult activity over the range of temperatures assessed was comparable to what was seen for *A. glabripennis* [22,23]. Adults held at 15–25 ◦C exhibited the full range of normal behaviors (resting, feeding, mating, and ovipositing), just more slowly at cooler temperatures. At 10 ◦C Chinese adults moved very slowly, fed little, and did not chew oviposition sites. At 30–35 ◦C adult behavior increased, and they would attempt to walk or fly away when their twigs and bolts were changed. At 35 ◦C, Chinese females did not oviposit even though they chewed some oviposition pits. Chinese adults held at 40 ◦C were more lethargic, fed little, and did not live long. The attempts to move away from the higher temperatures fit with the observations that *A. glabripennis* seeks shaded areas to rest when the temperature is >29 ◦C in China [24].

Adult longevity declined with the increase in activity at higher temperatures. This is likely due to increased metabolism at higher temperatures which has been found to reduce the life span in insects [25]. The longevity at 20–25 ◦C on *A. rubrum* for both sexes from the Chinese population is comparable to that recorded on *Melia azedarach* (91.8 days female and 103.0 days male) and higher than recorded on *Casuarina* sp. (61.6 days female and 63.0 days male) at room temperature in China [1]. Conversely, both sexes of the Italian population on *A. rubrum* at 25 ◦C lived for a shorter time than did individuals from a Japanese population at 24 ◦C on either *Citrus unshiu* (Yu.Tanaka ex Swingle) or *Salix schwerinii* E. Wolf [15]. This highlights the effects of host plants on longevity and suggests that caution should be used when applying predictions solely based on a single host in the laboratory. Female *A. chinensis* from both populations lived longer than *A. glabripennis* females at temperatures ≤15 ◦C, but comparable lengths of time at higher temperatures [23]. However, the male longevity of individuals from the Chinese population was comparable to that of *A. glabripennis* individuals over the 10–35 ◦C range [23]. The longer survival of female *A. chinensis* than *A. glabripennis* at cooler temperatures is surprising, since the native range of *A. glabripennis* extends further north than that of *A. chinensis*, but fits with the fact that *A. glabripennis* has a higher metabolic rate than *A. chinensis* at cold temperatures [17].

The timing of first oviposition (13–17 days to first egg at 24 ◦C depending on the host) and the shape of the weekly oviposition curve (bell-shaped with a long right tail) are comparable to the findings of this study for *A. chinensis* [1,7]. The mean fecundity (92 for Chinese and 24 for Italian at 25 ◦C), however, was lower than that of either Chinese females on *M*. *azedarach* (176 eggs) or field-reared Japanese females on citrus (193.8 eggs) [1,7]. This is most likely due to the host used in the studies, since the host has been shown to affect fecundity in *Anoplophora* spp. [15,26]. The increasing number of oviposition sites chewed per week, maximum oviposition per week, and fecundity for females from the Chinese population with increasing temperatures 15–30 ◦C was to be expected because of increased efficiencies of assimilation and conversion of ingested food at higher temperatures [27]. What was not expected was a lack of temperature response for reproductive output for the Italian population, and the causes for this are unknown. Compared to *A. glabripennis*, the *A. chinensis* Chinese females laid more eggs across the entire temperature range of 15–30 ◦C, and fecundity peaked at a higher temperature [23]. The higher fecundity could have been because *A. chinensis* were, on average, heavier than *A. glabripennis* females, and

fecundity has been correlated with size in both species [7,28]. The ability of *A. chinensis* to handle higher temperatures better than *A. glabripennis* is also suggested by its native range extending further south, even though much of their ranges overlap [2]. At the more extreme temperatures, the pattern of reproductive arrest seen for *A. chinensis* is comparable to that found for *Hylotrupes bajulus* L. [29] and *A. glabripennis* [28]. At 10 ◦C and ≥35 ◦C, females laid no eggs, and there were no fully developed eggs in their ovarioles at death. This was either due to a lack of egg maturation or eggs being reabsorbed within these females to use as an energy source.

Eggs of most insect species will hatch over a 15 ◦C range of constant temperatures, and under varying temperatures like occur in nature, egg hatch may occur 3–4 ◦C below that range [30]. As temperatures approach (within 2–3 ◦C), the *Tmax* and *Tmin*, percentage hatch declines dramatically [30]. *Anoplophora chinensis* eggs from the Chinese population appear to be an exception, as eggs hatched over a broader range of constant temperatures (15–35 ◦C), although the predicted *Tmin* and *Tmax* were only 3 ◦C below the temperatures at which hatch percentage declined sharply. However, the cold tolerance of *A. chinensis* eggs seems to be less than that of *A. glabripennis*, since none of the *A. chinensis* eggs that were held at 5–10 ◦C for 200 days and then were moved to 25 ◦C were able to hatch, while eggs of *A. glabripennis* did hatch under similar conditions. This would suggest that *A. chinensis* eggs would not be able to overwinter, unless the more sheltered tree bole and roots location they use provides more protection from colder temperatures than is afforded *A. glabripennis* eggs laid under the bark up higher on the tree. However, care should be taken in making this assumption, since removing the eggs from the bark can damage *A. chinensis* eggs, and we did not confirm this by holding eggs under the bark at the low temperatures.

The percentage of Italian eggs laid at 20 ◦C and hatched at 25 ◦C was similar to that for Japanese *A. chinensis* eggs laid and hatched at 20 ◦C in *Citrus* sp. Bolts (90%) [7]. The lower hatch percentages seen for eggs removed from bolts was probably due to damage sustained when removing them, since there were reductions for all temperatures, and the eggshells of *A. chinensis* broke more easily than those of *A. glabripennis*. The percentage hatch of the Chinese population eggs kept on bolts was comparable to *A. glabripennis* egg hatch when removed from bolts at 15–30 ◦C. However, unlike *A. glabripennis*, some *A. chinensis* eggs that were removed from bolts were able to hatch at 35 ◦C, possibly another indication that *A. chinensis* may be better adapted for warm climates than *A. glabripennis.*

The predicted number of degree-days to 50% hatch of *A. chinensis* eggs was 166 *DD* which is comparable to previous estimates for Japanese *A. chinensis* (184 *DD*) [7], but considerably less than the 239 *DD* required by *A. glabripennis* [23]. The *Tmin* of 12.7 ◦C is higher than the 6.7 ◦C estimated for Japanese *A. chinensis* and slightly higher than the 9.7 ◦C estimated for *A. glabripennis* [7,23]. In addition, the *Tmax* is quite a bit higher for *A. chinensis* than *A. glabripennis.* The lack of hatch for *A. chinensis* eggs held at 10 ◦C substantiates the *Tmin* estimated in this study and suggests that either the previous estimate was low or that some Japanese populations of *A. chinensis* may have a lower threshold.

Diurnal and seasonal temperature variations in the lower bole and exposed roots of trees where *A. chinensis* eggs are laid could be substantially different from ambient or from tree trunks and branches where *A. glabripennis* eggs are laid. Previous studies have shown that phloem temperatures in the upper bole and branches can be much higher than ambient (averaging 2–10 ◦C higher and maximum 21 ◦C higher), especially when the tree section is exposed to direct sunlight [31,32]. However, other factors can also alter within-tree temperatures, such as reflected and thermal radiation, wind chills, and soil temperature around the roots. In fact, the deeper in the trunk or under soil, the more insulated the wood is, and the slower the temperatures may respond to ambient changes [32]. When using the estimated *DD* and *Tmin* for predicting when *A. chinensis* eggs will hatch, ambient air temperatures will likely not provide accurate estimates. Further work to determine exactly how air temperature differs and temperature changes diurnally and seasonally in the root zone of trees is needed to accurately predict egg hatch timing and potential effects on percentage hatch for *A. chinensis.*

The impact of temperatures on the adults can be seen if the temperatures from the most northern and southern invasive *A. chinensis* populations in Europe are compared. Monthly mean temperatures in Rome, Italy are 17, 21, 23, 24, 21, and 17 ◦C during May, June, July, August, September, and October, respectively, while the corresponding temperatures in Rotterdam, Netherlands were 12, 15, 17, 17, 14, and 11 ◦C, respectively (https://weatherbase.com, accessed 2 April 2021). For Rome, Italy, during the same time period, the maximum temperatures averaged 22, 26, 29, 29, 26, and 22 ◦C, the maximum recorded temperature was 37 ◦C for July-September, and there are, on average, four days a year that the temperature exceeds 32 ◦C. Thus, Rome experiences temperatures at which the beetles can survive and reproduce well from June to August, and summer temperatures rarely reach >32 ◦C, so it should not reduce adult survival or suspend oviposition. Average temperatures in Rotterdam, however, barely reach levels at which beetles will oviposit June–August, and even high averages only reach ≥20 ◦C in July and August—so although the beetles may survive a long time, they will lay few eggs. This may partially explain the total of 25 exit holes seen in the Westland, Netherlands, infestation and the relative ease of eradicating that infestation, while the infestation in Rome is larger and more difficult to eradicate [11]. Average summer temperatures in major citrus-growing areas in California like Visalia would be near-optimal, but average highs and maximum recorded temperatures could suspend oviposition and reduce adult longevity part of the time. Citrus-growing areas in Florida (e.g., Bartow) would be near-optimum for beetles, and even the maximum recorded temperatures would rarely reach the *Tmax* for adult survival or result in the suspension of oviposition. These data on temperature effects on reproduction and survival of adults and eggs of *A. chinensis*, if combined with temperature response data for larvae, could be used to develop maps of the potential geographical range for this species and stage-based phenological models to predict the timing of life stages. Such information is critical for informing management and eradication programs, both in the native and introduced ranges.

### **5. Conclusions**

In the current study, the temperature had significant impacts on all the life-history traits of the Chinese population and most of the Italian population traits, with the exceptions of fecundity and mean maximum oviposition per week. Effects of temperature on the longevity of both sexes differed between the populations; Chinese individuals lived longer than Italian individuals. Also, adults from the Chinese population were heavier than those from the Italian population. Temperature effects on longevity also differed between the sexes. The percentage of females that oviposited and produced progeny was lower for females from the Italian than the Chinese population. There were no differences between the populations in the time to first oviposition or egg hatch rate over the temperature range that was evaluated. The mathematical relationships between temperature and the life-history parameters provided estimates of the lower and/or upper temperature thresholds and optimum temperatures for many of the traits evaluated. These relationships will facilitate the development of phenology models and improve predictions of potential geographic ranges to aid managers in deploying control and eradication measures.

**Author Contributions:** Conceptualization, M.A.K. and P.M.M.; methodology, all; formal analysis, M.A.K.; investigation, all; resources, M.A.K.; data curation, M.A.K. and G.B.; writing—original draft preparation, M.A.K.; writing—review and editing, all.; visualization, M.A.K.; supervision, M.A.K. and P.M.M.; project administration, M.A.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** The research was funded by the United Stated Department of Agriculture, Forest Service, Northern Research Station.

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** The data set from this study is deposited in the Forest Service data archive and the DOI is https://doi.org/10.2727/RDS-2021-0023 (last accessed 2 April 2021).

**Acknowledgments:** We thank Jessica Richards, Xingeng Wang, and the anonymous reviewers for their critical reviews of this paper that have helped improve it. Chris Fagan, Niklas Lowe, David Mikus, and Jessica Richards provided technical assistance. We also thank Nathalie Ramualde and Lincoln Smith of the USDA Agricultural Research Service for providing us with the individuals to start the colonies and advice on rearing.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


Author details

Daniela Domingos1 , Luis Felipe Lima1 , Thiago Messias1 , José Feijó1 , Anthony Diniz2 and Heliana Soares1 \*

1 Federal University of Rio Grande do Norte, Natal, Brazil

2 Federal University of Sao Paulo, Sao Paulo, Brazil

\*Address all correspondence to<EMAIL_ADDRESS>
© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


*3.3. Canyons*

The fishing targeting the deep-water shrimps in the northwestern Mediterranean is carried out on both the slope and the walls of the submarine canyons [11,14,67,120–124].

In the western Mediterranean, *C. conger* and *P. blennoides* are the fish species captured with the highest biomass at the head of the Blanes Canyon [124]. The juveniles of some deep-sea shrimps (e.g., *Plesionika heterocarpus, P. edwardsii, P. gigliolii*, and *P. martia*) and fish (such as *P. blennoides*, *Mora moro*, and *Lepidion lepidion*) are found to be distributed in the benthic intermediate nepheloid layers of the Blanes Canyon, which seems to act as a nursery area for these species [125,126]. From the canyons in the eastern part of the Gulf of Lions, blackmouth catshark and European hake are among the most abundant species [15]. Spawning females of angler and European hake have been more commonly observed within the submarine canyons of Petit-Rhône and Grand-Rhône than on the adjacent open slope [15]. *G. melastomus*, *H. dactylopterus*, and *P. blennoides* are the most frequently observed fish species in French Mediterranean submarine canyons characterised by the presence of CWC species [17]. Discarded fishing gear, including entangled nets and lines, has been observed in the canyons of the western Mediterranean where CWC species thrive [17,127–129].

The shark *G. melastomus* and the teleost fish *H. dactylopterus* and *P. blennoides* are the most frequently captured species in the Quirra Canyon (Sardinian waters), where valuable species—such as the European hake and the deep-water red shrimps—are also collected [130].

Eastward, in the Bari Canyon where CWC species thrive, the blackmouth catshark and the teleost fish European conger, blackbelly rosefish, European hake, blackspot seabream, and greater forkbeard have been found to be more abundant in the canyon than in the adjacent area [71,81]. In particular, greater forkbeard showed significantly greater abundance and biomass in the canyon than outside. Blackspot seabream are exclusively caught inside the canyon. A greater number of both smaller and larger individuals of European conger and greater forkbeard are found in the Bari Canyon than on the open slope. Mature females and males are mostly observed in the canyon in all of the most abundant species, indicating the role of the Bari Canyon as a refuge area and an EFH for fish species exploited in the neighbouring fishing grounds [71,83]. Longline remains have been observed in this canyon, which has a complex bottom topography and is not accessible to trawling [97].

Indeed, canyons seem to benefit and support fisheries [14,71,83,131,132], providing spawning and nursery sites as well as refuges for several commercial species [15,17,69,79, 80,84,97,122,126,133].


*2.2.2. Dynamic channel assignment schemes*

52 Wireless Mesh Networks – Efficient Link Scheduling, Channel Assignment and Network Planning Strategies

Channel Assignment (CA) in a multi-radio WMN environment consists of assigning channels to the radios in order to achieve efficient channel utilization (i.e. minimize cochannel interference) and, simultaneously, to guarantee an adequate level of connectivity. The problem of optimally assigning channels in an arbitrary mesh topology has been proven to be NP-hard, based on its mapping to a graph-coloring problem [11]. Therefore, channel assignment schemes employ heuristic techniques to assign channels to radios belonging to mesh nodes. A taxonomical classification of various CA schemes for wireless mesh

Fixed assignment schemes assign channels to radios either permanently, or for intervals that are long with respect to the radio switching time. Such schemes can be further subdivided

In CCA scheme [13], the radios of each node are all assigned the same set of channels. For example, if each node has two radios, then the same two channels are used at every node. The main benefit is that the connectivity of the network is the same as that of a single channel approach, while the use of multiple channels increases network throughput. However, it does not take into account the effect of interference on the channel assignment

In the VCA class of schemes, the radios of different nodes are assigned different sets of channels. However, the assignment of channels may lead to network partitions and/or topology changes, which may increase the length of routes between mesh nodes. Therefore, in such a scheme, channel assignment needs to be carried out carefully. The VCA approach is discussed in more detail by presenting algorithms that belong to this

C-HYA, a centralized channel assignment algorithm for multi-radio multi-channel WMNs, was proposed in [11]. Assuming that the offered traffic load is known, this algorithm assigns channels ensuring network connectivity and satisfying the bandwidth limitations of each

MesTiC, a fixed algorithm for centralized CA, was proposed in [14], and visits nodes once in the decreasing order of their rank. The rank of each node is computed on the basis of its

ii. Mesh-based Traffic and interference-aware Channel-assignment (MesTiC)

link-traffic characteristics, topological properties and number of radios on a node.

**2.2. Channel assignment schemes**

*2.2.1. Fixed channel assignment schemes*

**a. Common Channel Assignment (CCA)**

**b. Varying Channel Assignment (VCA)**

i. Centralized Hyacinth (C-HYA)

networks is as follows [12].

into two types.

in a WMN.

category.

link.

In dynamic CA schemes, any radio can be assigned any channel but additionally, radios can frequently switch from one channel to another. Therefore, when nodes need to communicate with each other in such a scheme, a coordination mechanism is required to ensure that they are on a common channel.

### **a. Multi-channel Medium Access Control (MMAC)**

MMAC [15] [16] is a link-layer multi-channel protocol for nodes with a single network interface. A node equipped with a single interface can only listen to one channel at a time. Therefore, in order to use multiple channels, the interface has to be switched between channels.

When nodes require to switch channels, a pair of nodes need to listen on the same channel at the time of communication and a channel coordination method is necessary, which is not required in TICA.

## **b. Distributed Hyacinth (D-HYA)**

D-HYA, a dynamic and distributed channel assignment algorithm proposed in [17], can adapt to traffic load dynamically. The algorithm builds on a spanning tree network topology. The gateway node is the root of the spanning tree, and every mesh node belongs to that tree. Based on per-channel total load information, a WMN node determines the set of channels that are least used in its vicinity. As nodes higher up in the spanning trees need more relay bandwidth, they are given a higher priority in channel assignment. The priority of a WMN node is equal to its hop distance from the gateway.

The CA schemes, such as C-HYA, MesTiC and D-HYA, require the traffic load to be known before assigning channels, whereas TICA requires no such knowledge for channel assignment.


**A Cost-Minimization Analysis of a Medical Record-based, Store and Forward and Provider-to-provider Telemedicine Compared to Usual Care in Catalonia: More Agile and E**ffi**cient, Especially for Users**

Received: 23 January 2020; Accepted: 17 March 2020; Published: 18 March 2020

**Abstract:** Background: Telemedicine (interconsultation between primary and hospital care teams) has been operating in the counties of Central Catalonia Bages, Moianès and Berguedà since 2011, specializing in teledermatology, teleulcers, teleophthalmology and teleaudiometries. For the period until the end of 2019, a total of 52,198 visits were recorded. Objective: To analyze the differential costs between telemedicine and usual care in a semi-urban environment. Methodology: A cost-minimization evaluation, including direct and indirect costs from a societal perspective, distinguishing healthcare and user's costs, was carried out over a three-month period. Results: Telemedicine saved € 780,397 over the period analyzed. A differential cost favorable to telemedicine of about € 15 per visit was observed, with the patient being the largest beneficiary of this saving (by 85%) in terms of shorter waiting times and travel costs. From the healthcare system perspective, moving the time spent in a hospital care consultation to primary care is efficient in terms of the total time devoted per patient. In social terms and in this context, telemedicine is more efficient than usual care. Conclusion: Allowing users to save time in terms of consultation and travel is the main driver of interconsultation between primary and hospital care savings in a semi-urban context. The telemedicine service is also economically favorable for the healthcare system, enabling it to provide a more agile service, which also benefits healthcare professionals.

**Keywords:** cost analysis; health technology assessment; provider-to-provider telemedicine; telehealth; economic analysis

**Francesc López Seguí 1,2, Jordi Franch Parella 3, Xavier Gironès García 3, Jacobo Mendioroz Peña 4,5, Francesc García Cuyàs 6, Cristina Adroher Mas 6, Anna García-Altés 7 and Josep Vidal-Alaball 4,5,\***


**1. Introduction**

The traditional production of small farmers in rural China has led to low agricultural production efficiency. In order to improve the efficiency of agricultural production, boost rural economy and achieve agricultural modernization in China, the Chinese government has been attaching great importance to encouraging the development of new agricultural operating entities, such as family farms. The Ministry of Agriculture issued the "Guiding Opinions on Promoting the Development of Family Farms" document in 2014, stating that the family farm mode is conducive to form moderate scale operations, which has played an important role in developing modern agriculture; therefore, it is of great importance to develop family farms in China. Subsequently, the No. 1 Central Documents, from 2015 to 2017, stipulated a series of measures to support the development of new agricultural operating entities, which created a nice social environment for the founding and developing of family farms. In 2019, the General Office of the State Council issued the "Opinions on Promoting the Organic Connection of Small Farmers and Modern Agriculture Development", which clearly proposed and clarified a family farm cultivation plan. The "High-Quality Development Plan for New Types of Agricultural Entities and Service Entities (2020–2022)", compiled by the Ministry of Agriculture and Rural Affairs in 2020, set the goal that the number of family farms in China will reach 1 million by 2022.

Under the guidance and support of government policies, the family farms in China have gained initial achievements, the number of which has been increasing every year.

**Citation:** Chen, Z.; Meng, Q.; Yan, K.; Xu, R. The Analysis of Family Farm Efficiency and Its Influencing Factors: Evidence from Rural China. *Land* **2022**, *11*, 487. https://doi.org/ 10.3390/land11040487

Academic Editors: Yongsheng Wang, Qi Wen, Dazhuan Ge and Bangbang Zhang

Received: 24 February 2022 Accepted: 24 March 2022 Published: 27 March 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

According to data from the Ministry of Agriculture and Rural Affairs of China, the number of family farms in China over quadrupled from about 139,000 in 2014 to 600,000 in 2018. However, although the number of family farms increased by 461,000 households, the growth rate declined year by year after 2015. The chain growth rate of family farms was initially 146.8% in 2015, followed by 29.7% in 2016, further decreased to 23.4% in 2017, and eventually fell to 9.29% in 2018. Another characteristic that should not be ignored is that most family farms in China are in a single type of planting or breeding operation. Among all family farms existing in any year between 2014–2018, the planting family farms account for the largest proportion, followed by breeding family farms. For example, in 2018, the planting, breeding, fishery, planting and breeding, and other types of family farms accounted for 62.7%, 17.8%, 5.3%, 11.6% and 2.6%, respectively, and similar distributions can be observed in other years.

Although we have witnessed an initial development of family farms in China, we have to admit that the total number of family farms is still minor. According to the data of the third agricultural census for China, the number of family farms only accounts for 0.2% of China's agricultural total households. In comparison to the traditional Chinese small farms, family farms have the characteristics of family-based production unit and scale operation [1], and they have been proved to be the most efficient mode in current agricultural production [2,3]. Therefore, it is of great significance to figure out how to improve the efficiency of family farms so as to maximize their functions in driving agricultural economic development. In order to promote the development of family farms, we have to answer the following questions: what is the actual operating efficiency of family farms in China? What are the factors that affect the efficiency of family farms? What are the differences in the efficiency and the influencing factors of family farms in different regions and different operation types?

### *1.1. Literature Review and Research Hypothesis*

#### 1.1.1. Family Farm Efficiency

Scholars have conducted a great amount of research on the efficiency of family farms. Many scholars confirmed that, through empirical analysis, the efficiency of family farms in China is not high [4–7], and the conclusions drawn from the decomposition of pure technical efficiency and scale efficiency varies. Qian and Li [4] conducted a DEA measurement and analysis of the efficiency of different types of family farms in Songjiang, Shanghai; they found that the efficiency of family farms is not high at 0.3841, and planting-andbreeding family farms have the highest efficiency compared to pure-grain-planting family farms and machine-farming family farms. Han et al. [5] argued that there are many DEA ineffectiveness units in 62 households of fruit-and-vegetable family farms in Zhejiang Province, and the average pure technical efficiency is greater than their scale efficiency. Li et al. [6] calculated that the technical efficiency of 234 family farms in Shandong Province was only 0.170, and the low value was attributed to the relatively insufficient pure technical efficiency rather than scale efficiency. The planting-and-breeding family farms had the highest efficiency, while pure-planting family farms were the least efficient. The research of Gao et al. [8] proved that the technical efficiency of family farms depends more on scale efficiency, while the pure technical efficiency is low.

#### **Hypothesis 1 (H1).** *The efficiency of family farms in China is low.*


**Abstract**

The creation of homozygous parental lines for hybrid development is one of the key components of commercial maize breeding programs. It usually takes up to 6 to 7 generations of selfing to obtain homozygous inbreds from the initial cross using the conventional pedigree method. Using doubled haploid (DH) method, concurrent fixation of all the genes covering entire chromosomes is possible within a single generation. For generation of DH lines, haploids are generated first by several means such as *in-vitro* method using tissue culture technique and *in-vivo* method using the haploid inducer (HI) lines. Of which, tissue culture-based methods have shown little promise for large-scale DH production as it needs good infrastructures and technical requirements. In contrast, inducer-based method provides more optimistic solutions for large-scale DH lines production. Due to its rapidity, DH technology is now being adopted in many countries including India for reducing the breeding cycle.

**Keywords:** doubled haploid, homozygous line, maize, and haploid inducer, inbreds

### **1. Introduction**

Maize breeding strategies rely heavily on the creation of homozygous parental lines for hybrid breeding. Using the traditional pedigree approach, it might take up to 7 generations of selfing to achieve homozygous inbreds from the first cross (**Figure 1**). In this context, because of its economic and logistical practicality, the creation of doubled haploid (DH) has received a lot of attention for varietal development in the last two decades [1]. The DH approach allows for simultaneous fixation of all genes across complete chromosomes in a single generation [2]. Haploids are initially created by a variety of methods, including an *in-vitro* way utilizing tissue culture techniques and an *in-vivo* method employing haploid inducer (HI) lines. On the other hand, Tissue culture-based approaches have not shown much promise for large-scale DH production since they necessitate adequate infrastructure and technical requirements. In contrast, because of its viability for large-scale DH line production, the inducer-based technique is deemed more hopeful and cost-effective [3]. According to the parent from which the haploids are being formed, inducer-based haploids may be divided into maternal haploid and

**Figure 1.** *Conventional breeding method for obtaining inbred lines.*

paternal haploid [1]. With the exception of their cytoplasm, the genetic constitutions of both forms of haploids are identical. Once haploids are created, their genetic constitutions can be duplicated in a single step to produce homozygous DH lines. DH technique has recently been implemented in commercial maize breeding programs in various countries because of its speed in decreasing the breeding cycle.


# Chapter 17

## Visualization and Functional Analysis of Spindle Actin and Chromosome Segregation in Mammalian Oocytes

### Binyam Mogessie

#### Abstract

Chromosome segregation is conserved throughout eukaryotes. In most systems, it is solely driven by a spindle machinery that is assembled from microtubules. We have recently discovered that actin filaments that are embedded inside meiotic spindles (spindle actin) are needed for accurate chromosome segregation in mammalian oocytes. To understand the function of spindle actin in oocyte meiosis, we have developed high-resolution and super-resolution live and immunofluorescence microscopy assays that are described in this chapter.

Key words Actin, Microtubules, Chromosomes, Meiosis, Oocytes, Eggs, Spindle, Fertility, Highresolution live microscopy

#### 1 Introduction

Every mammalian life begins with the fertilization of an egg by a sperm cell. For the resulting genetically unique zygote to grow into a healthy offspring, both the egg and sperm should first contain the correct number of chromosomes. However, for reasons we are only starting to understand, eggs are often likely to have additional or missing copies of certain chromosomes before fertilization—they are aneuploid [1]. Embryos formed from fertilization of aneuploid eggs frequently die, leading to pregnancy failures, or result in offspring with genetic disorders such as Down's syndrome. Importantly, the rate of egg aneuploidy increases dramatically with advancing maternal age [2]. This phenomenon, often referred to as "the maternal age effect," is highly attributed to errors in meiosis, the specialized form of cell division that generates eggs from oocytes [3]. Indeed, a number of factors including oocyte

The original version of this chapter was revised. The correction to this chapter is available at https://doi.org/ 10.1007/978-1-0716-0219-5\_22

Helder Maiato (ed.), Cytoskeleton Dynamics: Methods and Protocols, Methods in Molecular Biology, vol. 2101, https://doi.org/10.1007/978-1-0716-0219-5\_17, © The Author(s) 2020, corrected publication 2020

chromosome cohesion [4] and microtubule dynamics [5] are known to deteriorate with increasing maternal age.

Meiotic chromosome segregation is driven by a spindle machinery that is assembled from microtubules and separates the chromosomes in two rounds of cell division [3]. Interestingly, we recently discovered that the actin cytoskeleton plays a vital role inside the meiotic spindle—actin filaments embedded inside the spindle help to organize microtubules into functional bundles that can accurately separate the chromosomes [6]. This finding constitutes an important safety mechanism in mammalian meiosis that prevents aneuploidy in oocytes and eggs. The association of actin filaments with meiotic spindles of mouse oocytes has long been known [7, 8], but its functional analyses had been thwarted due to lack of high spatial and temporal resolution microscopy assays. This chapter discusses in detail the live and immunofluorescence microscopy assays that have for the first time enabled highly resolved visualization and functional analysis of spindle actin in mammalian oocyte meiosis.

#### 2 Materials

2.1 Ovary Dissection and Oocyte Isolation 1. N6,20 -O-dibutyryladenosine 30 ,50 -cyclic monophosphate sodium salt (dbcAMP). 2. 35 mm Tissue culture dishes. 3. Pastettes (e.g., Alpha Laboratories, LW4206). 4. Paraffin (mineral oil). 5. 37 -C incubator. 6. 1.5 mL microcentrifuge tubes. 7. Mouth aspirator tube assemblies for calibrated microcapillary pipettes (e.g., Sigma, A5177-5EA). 8. 100 μL micropipettes (e.g. PIP3022, Scientific Laboratory Supplies).

#### 2.2 Stock Solutions for Oocyte Culturing and In Vitro Maturation M2 Medium

2.3 mRNA Synthesis and Preparation for Microinjection

	- 2. Hydrochloric acid solution.
	- 3. Microwave.
	- 4. Absolute ethanol.
	- 5. Tungsten carbide scriber (glass cutter) (e.g., Micro-Mark, 50299).
	- 6. Microinjection chamber constructed from plastic according to dimensions specified in Subheading 3.2, step 2.
	- 7. Scotch double-sided tape, 12.7 7.9 mm.
	- 8. P-1000 micropipette puller fitted with a box filament (World Precision Instruments).
	- 9. Zeiss AxioVert A1 microscope equipped with Narishige MN-4, MO-202 U and NZ-19-2 micromanipulators, and a CellTram 4r Oil hydraulic manual microinjector (Eppendorf).
	- 10. Mercury.

2.5 High-Resolution and Super-Resolution Live Imaging, and Drug Addition Experiments

2.6 Immunofluorescence Microscopy of Fixed Oocytes

#### 3 Methods

3.1.1 Preparation of Oocyte Transfer and Washing Dishes

#### 3.1 Oocyte Isolation, Culturing, and In Vitro Maturation

Mouse ovaries are typically collected from 6- to 12-week-old females (e.g., 129 s, CD1, C57BL/6). Depending on the strain used, typically 30–50 oocytes can be obtained from each animal. This is normally sufficient for one live imaging or immunofluorescence experiment. Ovaries should be dissected and processed with as little delay as possible to maximize the number of meiosiscompetent oocytes that can be isolated. All culturing medium and dishes should be prepared and stored at 37 -C for prompt transfer of dissected ovaries. Oocytes should be cultured in dbcAMP (a non-hydrolyzable analog of cAMP) to keep them arrested in prophase during micromanipulation.

Fig. 1 Preparation of oocyte culturing and in vitro maturation dishes. Dishes should be prepared well ahead of oocyte isolation and microinjection experiments and stored at 37 -C

Fig. 2 Ovary dissection and puncturing.  (b) Mouse ovaries after extraction from surrounding fat tissue. (c) Intact (left) and punctured (right) mouse ovaries

3.1.2 Isolation and Preparation of Oocytes for Microinjection

Fig. 3 Mouth pipette production and assembly for aspiration of mouse oocytes.  (b) The process of cleaning isolated oocytes from debris by washing through nine droplets of M2 + dbcAMP. (c) Production of glass micropipettes for mouth pipetting using fire-assisted manual pulling techniques. Micropipettes with sharp or broken edges should be discarded to avoid oocyte death during mouth pipetting

Microinjection of oocytes is a principal method for studying mammalian meiosis by high-resolution live imaging. We routinely perform quantitative microinjection of oocytes using a modification of a method that was described in great detail by Jaffe and Terasaki [9]. In this section, technical details in the context of mouse oocyte microinjection are provided along with visual aids.

Visualization of spindle actin, microtubules, and chromosomes in live oocytes is achieved by microinjection of in vitro-transcribed mRNAs that encode their fluorescently labeled markers. We routinely use the calponin-homology domain of utrophin (UtrCH) to mark actin filaments, the microtubule-binding domain of MAP4 (MAP4-MTBD) to mark meiotic spindles, and histone H2B to mark chromosomes. To avoid degradation of mRNAs, they should be prepared for microinjection as follows only after isolation of oocytes from ovaries. In addition, all mRNA samples should be handled on RNase-free bench with gloved hands and using pipettes thoroughly cleaned with 70% ethanol.

3.2 Studying Spindle Actin Function Using High-Resolution and Super-Resolution Live Microscopy

3.2.1 Preparation of mRNAs for Microinjection

Fig. 4 Preparation of mRNA for microinjection. (a, b) Procedures to produce a small contraption to hold capillaries during mRNA loading.  (c) Stick folded capillary holder on a 25 75 mm glass slide using tape and place a dollop of grease to hold the capillary in place. (d, e) Load into the capillary the indicated volumes of oil, mRNA, and oil and place on capillary holder grease. (f) Place loaded capillary on 25 75 mm glass slide containing a dollop of grease. Transfer the capillary-containing slide into a 15 cm petri dish overlaid with tissue and place the petri dish on ice

11. Load 0.5 μL of capillary oil into the capillary and place it on ice inside the capillary storage dish by gently laying it over the grease dollop (Fig. 4d).

3.2.2 Preparation of Microinjection Chamber A U-shaped microinjection chamber can be constructed from clear plastic with little effort by most workshops according to the dimensions in Fig. 5a.  The cleanliness of coverslips used for microinjection strongly affects oocyte health. We use the following steps to thoroughly clean and store coverslips:

Fig. 5 Preparation of chamber and oocytes for microinjection. (a) Dimensions and sides of a microinjection chamber that can be constructed from plastic. (b) Cutting of coverslips into smaller pieces for construction of microinjection shelves. The non-cut (non-sharp) edge of shelving coverslips should be stuck on double-sided tape as an overhang to create space between the two coverslips. The side of the coverslip to which the shelf is affixed with double-sided tape is marked with asterisk symbol to aid with orientation. (c) Apply a thin layer of grease and mount a 22 22 mm coverslip on the back of the microinjection chamber. (d) Apply a thin layer of grease and mount a microinjection shelf on the front of the chamber. The shelving coverslip should face the medium dam. (e) Fill the dam space created on the chamber with M2 + dbcAMP. (f) Load oocytes into microinjection shelf by mouth pipetting. (g) Apply a thin layer of grease into the holding groove and mount mRNA capillary

We then prepare a microinjection shelf and chamber using the following steps:

(a) Using fine tweezers, place a double-sided tape roughly 5 mm away from either side of a 22 22 mm coverslip (Fig. 5b) and firmly stick it down by pressing on it using the non-sharp end of the tweezers.

Fig. 6 Positioning of oocytes, mRNA capillary, and chamber for microinjection. (a) Oocytes loaded into microinjection shelf by mouth pipetting. (b) Microinjection needle holder connected to hydraulic manual injector. (c) Needle, chamber, and mRNA-loaded capillary positioned on microinjection microscope

Fig. 7 Loading of microinjection needle with mRNA. (a–c) Steps of breaking the tip of the microinjection needle and moving mercury to the front of the needle. (d–k) Steps of needle loading with oil and mRNA

that the needle is not sufficiently broken. Repeat the tip breaking step and try to move the mercury to the front of the needle again.

Fig. 8 Microinjection of mouse oocytes. (a) Positioning of microinjection needle in focus with oocytes. (b) Microinjection needle in contact with oocyte at a focal plane that is not ideal for injection (above the hemispheric region of the oocyte). (c) Microinjection needle in contact with oocyte at the ideal focal plane. (d) Piercing into oocyte with microinjection needle followed by expulsion of oil and mRNA into the oocyte cytoplasm. When the front oil portion and mRNA volumes are expelled, mercury can be seen returning to the needle's front

the adjusted position, it should not be necessary to move the needle in the Z-axis throughout the rest of the microinjection experiment.

24. After microinjection, transfer oocytes to a prewarmed dish of M2 + dbcAMP droplets prepared earlier for mRNA expression.

For live imaging of meiosis in mammalian oocytes, we have routinely used confocal microscopes from Zeiss (LSM 710, LSM 780, LSM 800, LSM 880) and Leica (SP8). To achieve maximum resolution during imaging, we use 40 and 63 water immersion objectives (1.1–1.2 NA). In addition, we are able to achieve high signal-to-noise ratio using the Airyscan super-resolution module on LSM 800 and LSM 880 microscopes. Combined, these setups allow us to perform four-dimensional (x,y,z,t) live-cell imaging of meiosis without compromising oocyte viability.

To perform live imaging of microtubules, chromosomes, and spindle actin, oocytes must first be released from prophase arrest by washing out dbcAMP. Depending on the mouse strain used, the release from prophase arrest can take 30 min to 1 h. We find that oocytes from 129-s mouse strains release from prophase arrest faster than oocytes from CD1, C57BL/6, and FVBN strains. Achieving a temperature of 37 -C inside the imaging dish, close to the objective lens where oocytes eventually settle, is absolutely critical for oocyte development (see Note 18). To visualize the entire process of meiosis starting from nuclear envelope breakdown, oocytes should be transferred to the microscope immediately after washing out dbcAMP.

3.2.4 High-Resolution Live Imaging of Spindle Actin Assembly and Chromosome Segregation During Meiosis I and II

Fig. 9 Live imaging of spindle actin, microtubules, and chromosomes during mouse oocyte meiosis. (a) Single confocal section images of surrounded and non-surrounded nucleolar configurations of chromosomes (magenta) in prophase-arrested mouse oocytes. (b) Optimal signal-to-noise ratio of microtubules (gray) and chromosomes (magenta) in an oocyte-resuming meiosis after dbcAMP washout. (c) Spindle actin (white) and chromosomes (magenta) in a mouse oocyte progressing through meiosis I. The signal-to-noise ratio of spindle actin, cytoplasmic actin, and cortical actin indicates optimal mRNA expression level of EGFP-UtrCH that does not cause stabilization of actin filaments and perturb meiotic maturation. (d) Single-plane super-resolution (Airyscan) live imaging of actin (green), microtubules (gray), and chromosomes (magenta) in a mouse oocyte during spindle bipolarization. Faint labeling of actin filaments during spindle bipolarization indicates optimal expression of EGFP-UtrCH that will not interfere with spindle relocation and chromosome segregation due to actin stabilization

Fig. 10 High-resolution live imaging of meiotic spindle assembly and chromosome segregation in a mouse oocyte. Upon release from prophase arrest, a meiotic spindle is assembled from microtubules (gray). Homologous chromosomes (magenta) are captured by the spindle and transported to the oocyte surface where they are segregated. After a second meiotic spindle assembles and aligns the chromosomes, the egg is arrested in metaphase of meiosis II until fertilization

> conditions should allow continuous live imaging of oocytes from nuclear envelope breakdown through meiosis I to anaphase I and metaphase II spindle assembly without compromising oocyte health (Fig. 10) (see Note 23).

3.2.5 Pharmacological Drug Addition Experiments Cytoskeletal loss-of-function assays can be readily performed in oocytes by addition of widely used cytoskeletal inhibitors to the culture medium. Cytoskeletal disruption is reversible when oocytes are treated with drug concentrations we routinely use. We typically treat oocytes with 5 μg/mL cytochalasin D to disrupt the actin cytoskeleton, with 5 μM nocodazole to disrupt microtubules and 5 μM SiR-actin to stabilize actin. In addition, cytochalasin D and nocodazole can be combined to simultaneously disrupt actin filaments and microtubules in loss-of-function assays. Drugs can be added before releasing oocytes from prophase arrest. Alternatively,

Fig. 11 Cytoskeletal disruption assays for studying spindle actin function in mouse oocytes. Oocytes should be washed through 9 prewarmed 20 μL droplets of DMSO (control), cytochalasin D (to disrupt spindle actin), or nocodazole (to disrupt microtubules) before being placed in a glass-bottom imaging dish containing a droplet of DMSO or the respective cytoskeletal drug. For simultaneous drug treatment experiments, as many as four droplets can be placed in the imaging dish without risking cross-contamination

acute cytoskeletal disruption or stabilization can be achieved by addition of drugs at any desired stage of meiosis (e.g., spindle relocation, immediately before anaphase I or II, or before and after metaphase II spindle assembly). For analysis of spindle actin function in meiosis II chromosome alignment and segregation, cytochalasin D or SiR-actin should be acutely added at least 4 h after metaphase II spindle assembly to ensure that all chromosomes are fully aligned first.

3.2.6 Parthenogenetic Activation of Mouse Eggs to Visualize Spindle Actin and Chromosome Segregation During Anaphase II

6. Transfer oocytes to the imaging dish containing DMSO and drug droplets accordingly and proceed with microscopy.

7. If the experiment involves acute perturbation of spindle actin in meiosis II, perform the steps above with oocytes that have matured into eggs and have been arrested at metaphase II for at least 4 h (see Note 25).

After release from prophase arrest, mouse oocytes progress through meiosis I, segregate the homologous chromosomes in anaphase I, and become arrested in metaphase II until fertilized, when the sister chromatids are segregated in anaphase II. Importantly, fertilization can be chemically mimicked in vitro to induce anaphase II and observe sister chromatid separation. We follow these steps to efficiently achieve release from metaphase II arrest in mouse eggs:

3.2.7 Quantification of Chromosome Alignment and Segregation Errors from Live Imaging Datasets

Drug-mediated disruption of spindle actin or its genetic disruption in formin-2 knockout oocytes leads to chromosome alignment and segregation errors that lead to oocyte aneuploidy [6]. High temporal resolution is critical for quantification of chromosome misalignment and segregation. 3–6-min time-lapse live imaging datasets allow us to capture oocyte chromosome alignment and segregation in detail and to reproducibly quantify errors. These chromosomal defects can be quantified in two ways—firstly through manual quantification using well-defined criteria and secondly through automatic detection of chromosomes in live imaging datasets using Imaris software (Bitplane).

The first method takes into account the meiotic spindle length in maximum intensity projection confocal images to define outliers of chromosome alignment. As such, it should be used for quantification only in oocytes where the meiotic spindle is positioned parallel to the imaging plane (Fig. 12a). Z-projection of meiotic spindles that are oriented at various angles relative to the imaging plane will lead to inaccurate measurement of spindle length and the perception of chromosome misalignment where there is none (Fig. 12a). Alignment should be quantified in the metaphase frame immediately before anaphase onset. Anaphase onset is defined as the first frame where homologous chromosomes (anaphase I) or sister chromatids (anaphase II) noticeably start to move apart (Fig. 12b).

The second method uses the three-dimensional surface reconstruction module of Imaris software (Bitplane) to automate the detection of misaligned and lagging chromosomes from highresolution live imaging datasets. This approach can be used to independently confirm results from manual analyses. We have confirmed that the steps described here can be performed using Imaris versions 7.0–9.2.

#### 288 Binyam Mogessie

Fig. 12 Quantification of meiotic chromosome misalignment and segregation errors from high-resolution live imaging datasets. (a) Maximum intensity projections of microtubules (gray) and chromosomes (magenta) in a spindle that is parallel (left) and nonparallel (right) to the imaging plane. Because the spindle length appears shorter and the metaphase plate much wider, projection of nonparallel spindles gives the impression of chromosome misalignment where there is none, and thus should be avoided. (b) The frame of anaphase onset is determined as the time at which chromosomes (magenta) clearly begin to move toward opposite spindle poles. (c) Criteria for quantification of misaligned chromosomes and lagging chromosomes during meiosis are shown. Misaligned chromosomes are classified based on spindle length measurement as described in Subheading 3.2.7. Lagging chromosomes are defined as those chromosomes that fail to clear the central spindle region within 12 min of anaphase onset. Chromosomes that remain in the central spindle region 18 min after anaphase onset are classified as severely lagging. (d, e) Automated identification of misaligned (d) and lagging or severely lagging (e) chromosomes by three-dimensional iso-surface reconstruction of chromosomes (magenta) and using criteria described in (c)

For best results, mouse oocyte fixative solution should be prepared and prewarmed at 37 -C prior to fixation. In addition, the extraction and washing buffers should be prepared beforehand and stored at 4 -C when not being used.

#### 3.3 Studying Spindle Actin Function in Fixed Mouse Oocytes

3.3.1 Fixation and Preparation of Mouse Oocytes for Immunofluorescence

3.3.2 Labeling of Spindle Actin, Chromosomes, and Microtubules in Fixed Oocytes

#### 4 Notes

between imaging each color. Before nuclear envelope breakdown, only some acentriolar microtubule organizing centers (aMTOC) can be seen with MAP4-MTBD. UtrCH will produce a clear labeling of cortical actin and fainter labeling of cytoplasmic actin filaments, which will allow for later detection of spindle actin without stabilizing actin filaments (Fig. 9c). In meiosis-competent oocytes, chromosomes labeled with H2B-mRFP generally adopt a surrounded nucleolar (SN) configuration (Fig. 9a). In some oocytes, a non-surrounded nucleolar (NSN) configuration will be seen (Fig. 9a). Oocytes that achieve the surrounded nucleolar configuration before nuclear envelope breakdown are most likely to progress through meiosis. Since computing storage space is often a limitation with long-term live imaging of meiosis, it might be beneficial to only select those oocytes that have SN configuration for the overnight live imaging experiment. Although the microinjection techniques described here are highly quantitative and reproducible, some oocytes will often express more fluorescently labeled proteins than others. It is important to avoid imaging oocytes with much brighter fluorescence of actin, microtubules, and chromosomes as overexpression of each marker by itself will significantly perturb meiosis. In particular, overexpression of EGFP-UtrCH will stabilize actin filaments and prevent spindle relocation while overexpressed EGFP-MAP4- MTBD and SNAP-MAP4-MTBD will stabilize microtubules and lead to chromosome segregation errors. Similarly, overexpression of H2B-mRFP will interfere with chromosome individualization and segregation.

#### Acknowledgments

I would like to thank Kathleen Scheffler and Sam Dunkley for their input and critical reading of this manuscript. This work was supported by a Wellcome Trust and Royal Society Sir Henry Dale Fellowship.

#### References

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9. Jaffe LA, Terasaki M (2004) Quantitative microinjection of oocytes, eggs, and embryos. Methods Cell Biol 74:219–242

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